Effects Of Changes In Extracellular Calcium Biology Essay

Published:

The effect of extracellular calcium and magnesium ions on the contractile response to acetylcholine in the portal vein was the main aim of this study. Isolated portal veins from both rats and guinea pigs were suspended in an organ bath containing various compositions of krebs buffer solutions to which the different concentrations of calcium chloride, magnesium chloride and acetylcholine were added. Acetylcholine always produced a contractile response on the portal vein as activation of muscarinic receptor subtypes M2 and M3 by ACh in the tissue causes a contraction to be constantly induced, despite of whether depolarisation or hyperpolarisation of the membrane is occurring due to Ach. Calcium responded better to acetylcholine than magnesium, also producing larger responses. In Krebs buffer solution containing no calcium and in krebs buffer solution containing no magnesium, it can be seen that little or no effect is produced in the calcium free krebs buffer but an effect 7.5-fold greater in magnesium-free krebs solution, implying that contractions are dependent upon calcium as inward currents of a spontaneous nature along with influx of calcium via voltage-gated channels cause spontaneous contractions as they stimulate depolarisations. The portal vein didn't respond to acetylcholine in varying magnesium concentrations as well as it did for calcium because magnesium is potentially an antagonist to calcium thus preventing calcium influx, reducing contractile effects.

Acknowledgements

Lady using a tablet
Lady using a tablet

Professional

Essay Writers

Lady Using Tablet

Get your grade
or your money back

using our Essay Writing Service!

Essay Writing Service

I would like to thank Dr Munir Hussain for helping and guiding me throughout the 4 week duration of laboratory experiments, as well as during the preparation for the laboratory practicals. I would also like to show my gratitude and appreciation to the laboratory technicians Mark Filby and Richard Bottomley for providing us with the correct equipment in order to carry out the experiments and also their guidance throughout the practicals. I am also very grateful to Mark Filby and the fellow undergraduate students also present in the laboratory throughout the 4 weeks for making it an enjoyable working environment and an unforgettable experience.

Introduction

There are many distinctive features of the hepatic portal vein which differ from ordinary veins in other parts of the body. It has been previously demonstrated that mice's portal vein consist of a spiral fold structure which projects into the lumen (Barnett, 1952). Peristaltic movement of the hepatic portal vein has been demonstrated in vivo by Takahashi. Takahashi showed that in the trunk of the portal vein in which the spiral structure is recognised is only where contraction of the portal vein occurs. In the interval portion, longitudinal muscle cells contraction was demonstrated as during the portal vein's peristaltic movement, the interval of the spiral fold structure curtails.

Transmission of the longitudinal smooth muscle contraction occurs from the portal vein's mesenteric side to the hepatic side, therefore generating the vessel's peristaltic movement towards the liver (Takahashi et al., 2002).

Spontaneous phasic contractions can be seen in tissues of rat portal vein, which is shared by small resistance vessels. Spontaneous transient inward currents and calcium influx through voltage-gated channels are thought to be stimulating depolarisations which cause these spontaneous contractions (Burt, 2003). The perforation of chloride channels activated by calcium produce the portal vein's spontaneous transient inward currents (Pacaud et al., 1989; Wang et al., 1992). Evidence shows that in isolated tissues, these spontaneous contractions rely entirely upon extracellular calcium influx (Dacquet et al., 1987). If calcium is removed for a short period of time, rat portal vein contractions are eradicated, showing its entire dependency on external calcium (Marriott, 1988). This dependability of extracellular calcium has also been confirmed in numerous other studies but also that the spontaneous mechanical activity in the aforementioned vessels are inhibitorily effected by calcium-antagonistic drugs (Axelsson et al., 1967; Mikkelsen et al., 1984; Pegram and Ljung, 1981). Internal calcium does not notably contribute to and is not utilised for contraction of the portal vein (Axelsson et al., 1967; Marriott, 1988).

Stimulation of extracellular calcium influx, known as store-operated calcium entry, can occur due to calcium decreasing from internal stores in some portal vein cells (Burt, 2004). This store-operated calcium entry takes place through the smooth muscle's non-selective cation channels (Albert and Large, 2002). Internal calcium depletion can also have another effect in that depolarisation responses can be augmented as some depolarisations can result when non-selective cation channels open (Scharff and Foder, 1996). Gating of the non-selective cation channels can also occur in several different smooth muscle cell types due to M2 muscarinic receptor stimulation, playing an important role in excitation or contraction coupling (Kotlikoff et al., 1999).

Lady using a tablet
Lady using a tablet

Comprehensive

Writing Services

Lady Using Tablet

Plagiarism-free
Always on Time

Marked to Standard

Order Now

Increasing the free intracellular calcium concentration may cause contractions of vascular smooth muscle to commence but this can be due to calcium's intracellular stores mobilisation. It can also be due to membrane depolarisation activating voltage operated channels which have calcium influx from the external surroundings through them or through agonist-receptor combinations activating receptor-operated channels (Gregoire et al., 1993; Weiss, 1985).

Stimulated calcium influx in vascular muscle is prevented by organic calcium channel blockers (Flaim and DiPette, 1979). A variety of contractions dependent upon calcium in rat portal vein are inhibited by calcium entry blockers. Since an inward current of calcium triggers portal vein contraction, voltage-dependent slow calcium channels are likely to be the site of action of the entry blockers (Dacquet et al., 1987). However, external calcium entry via leak channels or calcium release from internal stores are not affected by these organic blockers (Flaim and DiPette, 1979).

Inositol 1,4,5 Triphosphate (IP3) levels are increased in response to stimulation of the portal vein's α1-adrenoceptors (Lepretre et al., 1994). Calcium released from cytosolic stores is stimulated by IP3 receptors (IP3R) bound to by IP3 which is produced by Phospholipase C after it is activated due to G-protein coupled receptors stimulation (Berridge et al., 2000). This release of internal calcium can initiate oscillations of IP3R-dependent calcium (Morel et al., 2003).

In vivo, it's clearly understood that any blood vessel contraction mechanisms are expected to differ, dependent upon the medical conditions, as they might affect calcium influx or cytosolic calcium release differently.

Increase in serum magnesium causes hypotension because of vasodilatation (Viveros and Somjen, 1968). A decrease is magnesium levels is often the cause of many disease that end in hypertension (Altura and Altura, 1982). Winkler suggested that if magnesium is externally introduced into patients suffering with renal and eclamptic hypertension, blood pressure will decrease (Winkler et al., 1942). It has been formerly proposed that by reducing permeability of the cell membrane and calcium's binding capacity, the excitation-contraction coupling in vascular smooth muscles can be affected by magnesium ions (Altura, 1970, 1975; Altura and Altura, 1971, 1974, 1976). Low concentrations of magnesium are dependent upon calcium for contraction but at higher magnesium concentrations, contractions are independent of extracellular calcium concentrations (Ohhashi and Azuma, 1982).

In vascular smooth muscle, it has been suggested that magnesium acts on several sites, one of which is the membrane. It is thought that the membrane is one of the sites acted upon for 3 reasons: 1) A rapid contraction is produced after a simple decrease is extracellular magnesium, 2) In the absence of extracellular magnesium, depolarised arteries' threshold for extracellular calcium-induced contraction is reduced, 3) Reduction in extracellular magnesium rapidly increases spontaneous mechanical activity whereas an rise in extracellular magnesium depletes this activity (Altura and Altura, 1974). Membrane permeability to external calcium is regulated by external magnesium or membrane sites are occupied by it, that are exchangeable with calcium bound to the membrane. Excitation of arterial smooth muscle resulting by inward movement through open specific membrane calcium sites by extracellular calcium result in external magnesium induced graded contractions (Altura and Altura, 1974).

Calcium release induced by calcium is enhanced by low magnesium, in striated muscle (Endo, 1977). In vascular smooth muscle, anions modulate calcium exchange and release by magnesium regulation, both intracellularly and in the plasma membrane (Zhang et al., 1991). Since some of calcium's vascular actions are opposed by magnesium, it has been suggested that magnesium could be a natural physiological antagonist of calcium (Altura et al., 1987).

Contraction and relaxation of arteries, veins and microvessels due to induction of drugs are affected by removal or decreasing external calcium (Altura and Altura, 1985; Altura et al., 1987). In vascular smooth muscle, some drug-evoked contractile responses can be split into fast and slow components (Bohr, 1964). These fast and slow components of the contractile responses are differentially affected by magnesium (Altura and Altura, 1974).

Within the guinea pig portal vein, depolarisation of the membrane, increase in ionic number and conductance, and frequency of spike generation occur due to Acetylcholine (ACh), meaning that the mechanical response of the portal vein is enhanced. ACh can also hyperpolarise the membrane and produce a contraction. Distribution on portal vein smooth muscle cell membrane of muscarinic receptors differ in properties than those in the main mesenteric vein since the portal vein membrane is depolarised by ACh but the main mesenteric vein membrane is hyperpolarised by it. Activation of muscarinic receptor subtypes M2 and M3 by ACh in longitudinal smooth muscle causes a contraction to be constantly induced, despite of whether depolarisation or hyperpolarisation of the membrane is occurring due to ACh (Bolton and Lim, 1991; Nanjo, 1984). In single cells, calcium-activated potassium channels are opened due to an increase in intracellular free calcium concentration caused by calcium released from stores induced by activation of muscarinic receptors. The aforementioned depolarisation of the membrane is caused by the opening of the previously mentioned channels, thus increasing the action potential discharge, causing contraction in the entire muscle (Bolton and Lim, 1991).

Lady using a tablet
Lady using a tablet

This Essay is

a Student's Work

Lady Using Tablet

This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.

Examples of our work

It has previously been shown that in rat portal vein myocytes, IP3R1 subtype expressed in cells by calcium transients and cells expressing both IP3R1 and IP3R2 are activated by calcium oscillations (Morel et al., 2003). Cytosolic calcium concentration regulation of IP3R2 is depended upon by calcium oscillations in vascular smooth muscle (Dupont and Combettes, 2006; Fritz et al., 2008). This is due to a change only in oscillating cells of ACh-induced calcium brought on by an increase in the concentration of internal calcium (Fritz et al., 2008). Fritz et al showed that IP3 oscillations aren't depended upon by ACh-evoked calcium oscillations where they express both subtypes IP3R1 and IP3R2 but rather they're dependent upon IP3R2's inherent sensitivity to internal calcium.

When given to some animals, Alloxan causes the destruction of pancreatic insulin producing cells causing Insulin Dependent Diabetes Mellitus (IDDM) therefore it is used to cause diabetes in laboratory animals for testing. The portal vein's sensitivity or responsiveness to noradrenalin, serotonin or potassium chloride does not change in rat portal vein with induced experimental diabetes (MacLeod and McNeill, 1985).

Aims and Objectives

The aim of this study is to isolate segments and measure contractility of the rat portal vein in order to assess the effect of extracellular calcium and magnesium ions on the response of contractile agents. Calcium chloride and magnesium chloride will be used to test the effects of contraction of the portal vein to acetylcholine, as well as alloxan. Another objective of this investigation is to compare the effects of the aforementioned aim on the rat portal vein and the guinea pig portal vein.

In this study, contractility will be measured of isolated portal vein to acetylcholine by addition of calcium and magnesium salts as the primary experiment. An isolated portal vein will be suspended in an organ bath containing krebs buffer solution to which the different concentrations of the calcium chloride, magnesium chloride and various drugs will be added. A dose response curve will be constructed from the resulting response graph from which conclusions will be derived.

Materials and Methods

Male adult Wistar rats/Dunkin-Hartley guinea pigs weighing between 250g - 350g were used during this experiment. They originated from Harlan UK Animal Research Laboratory. The rats were kept on a Harlan 2018 18% protein rodent diet, whilst the guinea pigs were kept on a Harlan 2040 guinea pig diet. They were fed Ad lib filtered tap water. The animals were housed in groups, bedded with Grade 6 woodchip and sizzle nest for the rats and hay for the guinea pigs. They were enriched with plastic and cardboard fun tunnels, plastic igloos and gnawing blocks. The animals were kept at room temperature (19-23°C) with a room humidity of 45-65%. They were exposed to 12 hours of lights and 12 hours of darkness everyday.

The animals were killed by cervical dislocation from which longitudinal strips (1cm in length, 0.5cm in width, and 0.5cm in depth) of the portal vein were isolated. These strips were suspended in an organ bath containing 20ml of Krebs buffer solution at 35°C, aerated with 95% O2 and 5% CO2. Using a thread, one end of the portal vein was tied to a metal tissue holder inserted into the organ bath, whilst the other end was tied to an ADInstruments force transducer in order for the tension to be measured.

The Krebs buffer solution was composed of (in mill moles (mM)): NaCl 118, NaHCO3 25, Glucose 11, KCl 4.7, CaCl2 2.5, KH2PO4 1.18, and MgSO4 1.18. A Ca2+-free Krebs solution was used sometimes of the same composition but with CaCl2 omitted. Alternatively, an Mg2+-free Krebs solution was also used, again of the same composition but with MgSO4 omitted. A Ca2+ and Mg2+-free Krebs solution was used sometimes of the same composition but with the omission of CaCl2 and MgSO4. A high-Ca2+ and Mg2+ Krebs solution was used, with the same composition of normal Krebs but with a concentration of 5mM for CaCl2 and 2.35mM for MgSO4. The portal veins were suspended in the organ bath and connected to the transducer so that they had a resting tension of between 1-1.4g. These changes in tension were detected by the force transducer and instantly recorded on graphs by ADInstruments Chart 5 for Windows.

Experiments in normal Krebs solution. Once suspended in the organ bath containing 20ml Krebs buffer solution, the portal veins were left between 5-10 minutes to look for any oscillations on the graphs, after which 0.2ml of 1 x 10-3 M ACh was added, resulting in a final bath concentration of 1 x 10-5 M for ACh. After this addition, any response to the drug was noted as a response to 2.5 x 10-1 M Ca2+ and 1 x 10-1 M Mg2+ as the Krebs buffer solution contained that specific concentration of the two chemicals. The buffer solution was cleared out of the organ bath and 20ml of fresh Krebs added. At this point, each one of the following volumes of 1 x 10-1 M Ca2+ were added to the organ bath; 0.2ml, 0.4ml, 0.6ml, 0.8ml or 1.0ml giving a final bath concentration for Ca2+ of 3.5 x 10-3 M, 4.5 x 10-3 M, 5.5 x 10-3 M, 6.5 x 10-3 M and 7.5 x 10-3 M respectively. The oscillations, if any, were recorded on the graph, before the addition of 0.2ml 1 x 10-3 M ACh, after which the response was recorded on the graph. In place of Ca2+, Mg2+ was also used by means of the same procedure but giving a final bath concentration for the aforementioned added volumes of 2 x 10-3 M, 3 x 10-3 M, 4 x 10-3 M, 5 x 10-3 M and 6 x 10-3 M respectively. The experiment was also repeated using both Ca2+ and Mg2+ via the same procedure for Alloxan in place of ACh, using the 0.2ml as well as 1.0ml and a concentration of 1 x 10-1 M. This concentration gave a final bath concentration for Alloxan of 1 x 10-3 M and 5 x 10-3 M for both the 0.2ml and 1.0ml volumes used respectively.

Experiments in Ca2+-free Krebs solution. The same procedure was used as for normal Krebs solution but only 0.6ml Mg2+ (1 x10-1 M, Final Bath Concentration: 4 x10-3 M) was used along with the stated ACh dose (no Ca2+ or Alloxan).

Experiments in Mg2+-free Krebs solution. The same procedure was used as for normal Krebs solution but only 0.6ml Ca2+ (1 x10-1 M, Final Bath Concentration: 5.5 x10-3 M) was used along with the stated ACh dose (no Mg2+ or Alloxan).

Experiments in Ca2+ and Mg2+-free Krebs solution. No additional Ca2+ or Mg2+ was added to the buffer solution, so only ACh was added as stated in the procedure for normal Krebs solution.

Experiments in high-Ca2+ and Mg2+ Krebs solution. No additional Ca2+ or Mg2+ was added to the buffer solution, so only ACh was added as stated in the procedure for normal Krebs solution.

Drugs and solutions. ACh, Alloxan and CaCl2 were obtained from Sigma. MgCl2 was obtained from Fisher Scientific. Distilled water was used to make all the stock solutions. All stock solutions were freshly made everyday and were accurate to ±0.2ml. ACh and Alloxan were kept on ice throughout the day.

Data Analysis. The contractions were measured as a response to ACh (1 x 10-5 M) by calculating the integral relative to the baseline of the raw data chart using LabChart 7 Reader and the mean calculated from 5 individual experiments (n = 5). From this, Dose Response curves were constructed. An independent t-test was used on raw data to test the significance of differences for statistical purposes where a P value of ï‚£0.05 was used. Statistical analysis was carried out using IBM SPSS PASW Statistics 18.

Results

All of the phasic contractions were measured by calculating the integral above the baseline as a response to ACh (1 x 10-5 M) for each tissue after the addition of different concentrations of calcium or magnesium (where required). This is calculated by the software by subtracting the value on a baseline from the sum of the data points on a specified area on the graph, which is then multiplied by the sample interval, for example, Figure 1 below shows an example of a selection (black highlight) which was used by the software to calculate the integral relative to the baseline after the addition of a final bath concentration of 1 x 10-5 M ACh. Prior to this, a final bath concentration of 7.5 x 10-3 M CaCl2 was added. The integral value given here was 20.6124 grams multiplied by seconds (g.s).

Tension 1g

Time - 1 minuteFigure 1. Contractile response to ACh (1 x 10-5 M)

selection to calculate integral above baseline.

Experiments in normal Krebs solution. The largest individual as well as average response to ACh was produced at 7.5 x 10-3 M of Ca2+ in the rat portal vein, with the largest individual response being 21.7879 g.s and the largest mean response being 20.45482 g.s. At a Ca2+ concentration of 6.5 x 10-3 M, the mean response was almost halved (10.92036 g.s). The response decreased to 8.14884 g.s at 5.5 x 10-3 M Ca2+, then rising to 12.78344 for 4.5 x 10-3 M Ca2+, the second highest mean response. At a Ca2+ concentration of 3.5 x 10-3 M, the response slightly decreased to 11.37472 g.s, then increasing to 12.4481 g.s in Krebs solution on its own (2.5 x 10-3 M Ca2+). The biggest variation from its mean response is for 4.5 x 10-3 M Ca2+, with a standard deviation (SD) of 4.5 g.s. A Ca2+ concentration of 7.5 x 10-3 M had a SD of 0.87 g.s, which shows the smallest variation from the mean response, in this case being 20.45482 g.s. All of the mean responses and SD have been represented in a Dose-Response curve (Figure 2).

At the highest Mg2+ concentration (6 x 10-3 M), the mean response (10.88362) was similar to that of 6.5 x 10-3 M Ca2+, and a SD of 1.76 g.s. The lowest response produced was at 4 x 10-3 M Mg2+ (5.85 g.s). The largest SD value was given at a concentration of 2 x 10-3 M of Mg2+ (2.77 g.s), varying from the mean of 10.86862 g.s. The highest mean response was at an Mg2+ concentration of 1 x 10-3 M of 12.4481 g.s (SD = 1.26 g.s). The highest individual response was 15.0248 g.s at 2 x 10-3 M Mg2+. The remaining responses and SD's, along with the aforementioned ones, are represented in a Dose-Response curve (Figure 2).

Figure 2: Dose-Response curve showing how different concentrations (means) of Ca2+ and Mg2+ affect the rat portal vein's contractile response to ACh.

Due to the guinea pig portal vein not responding to majority of different concentrations of both Ca2+ and Mg2+, dose response curves were not been constructed, but the remaining obtained data was used to compare mean response to ACh in both animals, as well as to compare the response of both Ca2+ and Mg2+ on rat and guinea pig (from the results obtained) portal vein's separately. It was also used to compare the responses of Ca2+ & Mg2+-free Krebs solution and high-Ca2+ & Mg2+ Krebs solution as data was obtained for this from the guinea pigs portal vein.

Independent t­-tests were carried out on the data to statistically analyse to see if variances were equal (H0) or different (H1) in rats and guinea pigs as well as in calcium and magnesium where a P value indicating a difference of statistical significance of 0.05 was considered. All of the responses to ACh, in terms of integral above baseline, each taken from 50 different experiments (n=50) of different concentrations of Ca­­2+ and Mg2+ on both rat and guinea pig portal veins give mean values in close proximities of each other. A mean integral of calcium and magnesium giving 11.66 g.s and 10.61 g.s is shown respectively, which are not too dissimilar but showing that the rat portal vein responds slightly better to ACh from varying Ca2+ concentrations than varying Mg2+ concentrations. The standard deviation of the two has a slightly bigger margin, with Ca2+ having a standard deviation of 5.24 and Mg2+ of 3.47 (Figure 3A). Levene's Test for Equality of Variances showed P = 0.025 < 0.05 suggesting that equal variances are not assumed. The 2-tailed test calculates the significance of the hypotheses on average. This gives P (2-tailed) = 0.243 > 0.05, meaning P (1-tailed) = 0.1215 (Figure 3B). This suggests that on average, the Integral of Calcium and Magnesium are equal.

For the rats and guinea pigs t-test, all Ca2+ and Mg2+ concentrations used on each animal were included. The means values are very close, with 10.43 and 10.35 respectively. The standard deviation of the two is also very close, with 4.35 for rats and 4.79 for guinea pigs (Figure 3C). Figure 3D shows P (1-tailed) = 0.4625 > 0.05 giving strong evidence that on average, the Integral of Rat and Guinea Pigs are equal.

Experiments in Ca2+ and Mg2+ free Krebs solution. The largest individual response came from the rat portal vein (9.4838 g.s), which also had the biggest mean of 6.89 g.s over the guinea pig portal vein (6.04 g.s) suggesting that the rat responds better to ACh than the guinea pig. The response for the rat varies from the mean at almost 2 g.s (1.95 g.s), but the guinea pigs SD is lower, at 1.65 g.s.

Experiments in high Ca2+ and Mg2+ Krebs solution. The rat portal vein produced the largest individual response of 10.6954 g.s as well as a higher mean average of 8.97684 (SD = 1.67) over the guinea pig portal vein's mean response to ACh of 5.16158 (SD = 3.00). This suggests that the rat portal vein responds to ACh in high Ca+ and Mg2+ Krebs solution a lot better than the guinea pig portal vein.

An independent t-test was also carried out for the comparison between Ca2+ and Mg2+-free Krebs solution with high-Ca2+ and Mg2+ Krebs solution. The means of the two types of Krebs solutions have a different of only 0.6 g.s and a standard deviation difference of 1.29 (Figure 3E). P (1-tailed) = 0.297 > 0.05, showing that the Integral above baseline as a response to ACh of Ca2+ & Mg2+-free Krebs solution and high-Ca2+ & Mg2+ Krebs solution are equal on average (Figure 3F).

A

Group Statistics

Chemical Element

N

Mean

Std. Deviation

Std. Error Mean

Integral (g.s)

Calcium

50

11.657494

5.2375898

.7407070

Magnesium

50

10.612604

3.4663039

.4902094

B

Independent Samples Test

Levene's Test for Equality of Variances

t-test for Equality of Means

F

Sig.

t

df

Sig. (2-tailed)

Integral (g.s)

Equal variances assumed

5.180

.025

1.176

98

.242

Equal variances not assumed

1.176

85.015

.243

C

Group Statistics

Animal

N

Mean

Std. Deviation

Std. Error Mean

Integral (g.s)

Rat

80

10.434621

4.3520224

.4865709

Guinea Pig

40

10.351965

4.7907393

.7574824

D

Independent Samples Test

Levene's Test for Equality of Variances

t-test for Equality of Means

F

Sig.

t

df

Sig. (2-tailed)

Integral (g.s)

Equal variances assumed

2.718

.102

.095

118

.925

E

Group Statistics

Concentration

N

Mean

Std. Deviation

Std. Error Mean

Integral (g.s)

Ca & Mg Free

10

6.465130

1.7607736

.5568055

High Ca & Mg

10

7.069210

3.0480988

.9638935

F

Independent Samples Test

Levene's Test for Equality of Variances

t-test for Equality of Means

F

Sig.

t

df

Sig. (2-tailed)

Integral

(g.s)

Equal variances assumed

3.944

.062

-.543

18

.594

Figure 3. A: Mean, Standard Deviation and Standard Error of Mean for all experiments of all concentrations of calcium/magnesium. B: Independent t-test for equal variances following on from Figure 2A between calcium and magnesium. C: Mean, Standard Deviation and Standard Error of Mean for all experiments carried out on rat/guinea pig. D: Independent t-test for equal variances following on from Figure 2C between rat and guinea pig. E: , Standard Deviation and Standard Error of Mean for all experiments carried out in Ca2+ & Mg2+-free Krebs solution and high-Ca2+ & Mg2+ Krebs solution. F: Independent t-test for equal variances following on from Figure 2E between Ca2+ & Mg2+-free Krebs solution and high-Ca2+ & Mg2+ Krebs solution.

Experiments in Ca2+-free Krebs solution. Out of the 5 different experiments tested, all 5 produced a response, so n = 5. The mean response was 1.482 g.s (SD = 0.57 g.s). The largest response produced was 1.9035 g.s.

Experiments in Mg2+-free Krebs solution. All 5 different experiments performed in this particular Krebs solution produced a response, so n = 5. The largest response to ACh produced was 14.4246 g.s, with an average of 11.22466 (SD = 3.428052).

By comparing both the experiments in Ca2+-free Krebs solution and Mg2+-free Krebs solution, it is evident that Ca2+ is required to achieve a contractile response to ACh, as the Mg2+-free Krebs solution contained 2.5 x 10-3 M Ca2+ whilst there was no Ca2+ in the other Krebs solution. Experiments in the Mg2+-free Krebs solution produced a contractile response 7.5-fold greater than those in the Ca2+-free Krebs solution. However, the variation from the mean response for experiments in Mg2+-free Krebs solution (3.43 g.s) is 6-fold greater than that for experiments in Ca2+-free Krebs solution (0.57 g.s).

Figure 4. Response of rat portal vein to Ach in experiments containing Ca2+-free Krebs and Mg2+-free Krebs.

Experiments in response to Alloxan. Alloxan had no affect on either the rat or guinea pig portal, on either 2.5 x 10-3 M, 3.5 x 10-3 M or 7.5 x 10-3 M Ca2+ as well as no affect on 1 x 10-3 M, 2 x 10-3 M or 6 x 10-3 M Mg2+, therefore there is no data to present for this particular experiment.

Discussion

This study has confirmed that the portal vein contracts better to ACh in varying calcium concentration than it does to varying magnesium concentrations. Also shown in this investigation is that there is little or no response in the absence of calcium compared to the presence of calcium. The present research also shows that both the rat and guinea pig portal veins respond to ACh through spontaneous tonic contractions but the rat portal vein responds more readily than the guinea pig portal vein. These findings also suggest that Alloxan has no affect on the portal vein.

The portal vein responds to ACh through tonic contractions because the response of the tissue is improved as ACh causes the membrane to depolarise, the ionic number is increased along with the conductance, and the frequency at which the spikes are generated is also enhanced. A contraction is then produced as the membrane is hyperpolarised by ACh. Muscarinic receptors M2 and M3 are distributed on the portal vein smooth muscle cell membrane, whether or not the membrane is being depolarised or hyperpolarised due to ACh, of which activation of these receptors causes a continuous contraction to be generated (Bolton and Lim, 1991; Nanjo, 1984). The contraction in the whole muscle is caused by opening of calcium-activated potassium channels which results in depolarisation of the membrane. These channels open from an increase in internal free calcium concentration, causing release of calcium from stores induced by muscarinic receptor activation (Bolton and Lim, 1991).

The portal vein responded very well to the different calcium concentrations because transient inward currents of a spontaneous nature along with influx of calcium via voltage-gated channels cause spontaneous contractions as they stimulate depolarisations (Burt, 2003). The spontaneous transient inward currents are caused by calcium causing the activation of chloride channels which then perforate (Pacaud et al., 1989; Wang et al., 1992). These contractions are totally reliant upon extracellular calcium influx (Dacquet et al., 1987). Contractions can also be due to receptor-operated channels activated by agonist-receptor combinations (Gregoire et al., 1993).

Depending upon the medical condition of the rat or the guinea pig, influx of calcium or cytosolic calcium release can be affected differently, therefore the mechanisms of contractions of the portal vein, as well as any other blood vessel, could be different.

Contractile responses in vascular smooth muscle caused by drugs are split into slow and fast components which are affected by magnesium contrastingly (Altura and Altura, 1974; Bohr, 1964). One of the reasons why the portal vein doesn't respond as good to magnesium than it does to calcium is because hypotension is caused due to vasodilatation when serum magnesium levels are increased (Viveros and Somjen, 1968). Reduction in the cell membrane permeability and the binding capacity of calcium, can cause magnesium ions to affect the vascular smooth muscle's excitation-contraction coupling (Altura, 1970, 1975; Altura and Altura, 1971, 1974, 1976).

It can be seen that at lower magnesium concentrations, the tissue responds better than at higher magnesium concentrations. This could be due to the fact that at lower concentrations, there is dependency from magnesium on calcium for the production of contractions. At higher concentrations, that dependency is reduced (Ohhashi and Azuma, 1982) with still being able to produce contractions, but not as large as those produce at lower concentrations. This suggests that calcium influx into the portal vein is reduced at higher concentrations of magnesium, thus producing these smaller contractions. This observation has previously been supported by Altura and Altura, where they suggested that magnesium acts on the membrane for three main reasons; production of a rapid contraction after a decrease in extracellular magnesium, contractions induced by external calcium shows reduced threshold of depolarised arteries when extracellular magnesium is absent, and when external magnesium is decreased the activity of the tissue rapidly increases but a rise in magnesium decreases this activity (Altura and Altura, 1974). Inward movement of extracellular calcium through open specific membrane sites of calcium caused by smooth muscle excitation causes contractions induced by external magnesium (Altura and Altura, 1974).

The vascular actions of calcium are opposed to the vascular actions of magnesium, suggesting that magnesium could possibly have natural physiological antagonistic effects on calcium (Altura et al., 1987) which also explains why at higher concentrations of magnesium the response of the portal vein was reduced when compared to lower magnesium concentrations, as the lower concentrations of magnesium weren't strong enough to have antagonistic effects as the higher concentrations would.

In the absence of calcium, there was scarcely a response produced in comparison to when calcium was present. In calcium's absence, when drugs are induced, contraction and relaxation of veins are altered (Altura and Altura, 1985; Altura et al., 1987). As mentioned previously, spontaneous contractions rely entirely upon extracellular calcium influx (Dacquet et al., 1987). The dependency of calcium on contractions has been shown previously as the contractions of the portal vein are non-existent when calcium is removed (Marriott, 1988; Pegram and Ljung, 1981). Very minor contractions were produced in the absence of calcium in this study, but this could have been due to very small amounts of calcium still circulating in the portal vein at the time of testing. Although intracellular calcium may have been present in the portal vein at the time, it doesn't contribute nor is it utilised for contractions of the tissue (Axelsson et al., 1967; Marriott, 1988).

The rat portal vein responded more readily to ACh for both calcium and magnesium than the guinea pig portal vein, which failed to contract to the majority of the different concentrations. At the beginnings of the experiments, the guinea pig portal vein responded, but as the days went on, the animals gained weight, and the guinea pig portal vein's responses decreased day by day. At the beginning of the experiments, the animals weighed around 250g. Towards the end of the experiments (approximately 4 weeks later), the animals weight around 350g, a 100g difference from the animals at the start to the animals at the end. As the guinea pigs gained weight, the liver gained weight therefore causing predominant post-sinusoidal constriction (Shibamoto et al., 2004) thus the portal vein not responding to ACh the way it should.

Alloxan caused no response to be produced when added to the portal vein. Contractions were abolished, which could suggest that alloxan is an antagonist of calcium, potentially causing the same effects as magnesium's antagonism on calcium in that it could prevent calcium influx, preventing contractions. It has previously been reported that the portal vein of alloxan induced diabetic rats show no affect in the response on contraction or the uptake and distribution of calcium throughout portal venous smooth muscle (Turlapaty et al., 1980a; Turlapaty et al., 1980b). The sensitivity of the portal vein to alloxan and calcium in different stages of diabetes was shown to decrease which could be caused by alterations in the metabolism of calcium in the portal vein smooth muscle cells in the diabetic state (Turlapaty et al., 1980b).

In order to further this study, the effect of calcium channel blockers on the contractile response of the portal vein to ACh can be tested. Calcium channel blockers include nifedipine, verapamil and diltiazem (Dacquet et al., 1987; Marriott, 1988). The effects of intracellular, as opposed to extracellular, calcium and magnesium can also be tested on the contractile response to ACh of the portal vein. In this investigation, the concentration of ACh was kept constant throughout, so testing the effects of higher or lower concentrations is another option. This demonstration tested the portal vein of rats and guinea pigs only. Further demonstrations could be carried out on rabbits, dogs and cats to compare the effects of this same study, along with the ones aforementioned on portal veins of different mammals.

In conclusion, this study shows that in different calcium concentrations, the portal vein's contractile response to ACh is better than that of differing magnesium concentrations due to calcium influx and spontaneous transient inward currents causing contractile activity, as well as magnesium acting as a potential antagonist to calcium thus reducing the response of the tissue to the varying concentrations of magnesium by reducing calcium influx. Calcium absence produces a parsimonious response in contrast to calcium presence, confirming previous observations that contractions are totally reliant upon extracellular influx of calcium. The portal vein failed to have any sort of response to alloxan, as it could potentially be an antagonist to calcium. The rat has a better contractile response to ACh than the guinea pig, the reason being that the guinea pigs increase in weight caused post-sinusoidal constriction.