Amylase In Pancreatic Acinar Cells Biology Essay

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ABSTRACT

The present investigation was to study the role of calcium on the exocrine pancreatic amylase secretion. This was carried out using two major agonist's cAMP and carbachol. The amylase activity was determined by measuring the concentration of radioactive maltose that was produced in response to these agonists. This was also measured in the presence of three different buffers; EGTA (no calcium), buffer + 1mM calcium and buffer + 4µM calcium. The results attained showed a high increase in the concentration of maltose in response to cAMP and carbachol individually as well as together. These results indicated that intracellular calcium was involved in the secretion of amylase via intracellular signalling pathways such as adenylyl cyclase pathway and the phospholipase C pathway which entails the stimulation of PIP₂ which is converted into IP₃ and DAG. IP₃ then goes on to release intracellular stores of calcium thus activating the secretion of digestive enzyme amylase.

INTRODUCTION

The exocrine pancreas plays a fundamental role in the secretion of amylase. This may occur through several pathways such as the cAMP pathway, IP₃ pathway and other none IP₃ and PIP₂ linked pathways. Agonists such as Acetylcholine (Ach) a neurotransmitter and Cholecystokinin (CCK) a hormone activate phospholipase C through muscuranic receptors (Boron and Boulpaep, 2009) which are found on acinar cells (Metz et al, 1992).

Amylase is an active substance when it is liberated, therefore we can measure the activity of it. It can then be used as an indicator of acinar cells to observe the amount of amylase secreted from them, in this experiment the amount of maltose was measured to calculate amylase activity. The acinar cells have two major pathways which are involved in the increase of intracellular free calcium. The release of calcium in the acinar cells is initiated by acetylcholine and cholecystokinins which together stimulate phospholipase C; this then stimulates Protein Kinase C. The other is calmodulin, which responds to an increase in calcium and therefore promotes the activation of protein kinases and proteases (Boronet and Boulpaep, 2009).

Calcium can enter and be stored in the pancreatic acinar cell by the means of cGMP.

This occurs due to cholecystokinin or acetylcholine as they elevate the levels of intracellular cGMP. This rise in cGMP occurs due to nitric oxide which was stated by (Boronet and Boulpaep, 2009). Carbachol is an agonist that activates the acetylcholine receptors, which was used in this experiment to measure the extent of calcium in response to amylase activity.

There are several ways in which intracellular calcium can be released from the stores which are contained within the endoplasmic reticulum. One of which is adenylyl cyclase which promotes cAMP formation and another is phospholipase C; this involves the conversion of phosphatidylinositol (4, 5) bisphosphate (PIP₂) into inositol 1, 4, 5 trisphosphate (IP₃) and diacylglycerol (DAG) this occurs via GPCR's (Rang & Dale, 2007).

G Protein Couples Receptors (GPCRs) are receptors which have a seven membrane spanning domain. They are located on the plasma membrane and when these receptors are activated, systems within the cell are stimulated to produce second messengers IP₃ and DAG. The role of G proteins, involves the binding of nucleotides to the alpha subunit which in turn causes the alteration of GDP to GTP. Then a GTP-alpha complex if formed which subsequently detaches from the receptor and binds to a protein which causes the activation of that protein; which in this case would be PLC (Rang & Dale, 2007). Adenylyl cyclase is controlled by the activation of GPCR's which could be the muscuranic receptors in this experiment. This activation causes the adenylyl cyclase to either increase or decrease the concentration of cAMP thus activating protein kinases such as protein kinase A (Rang & Dale, 2007).

The aim of this experiment was to see the effect of amylase activity in response to calcium in the presence of different agonists. Calcium, cAMP and CCh were used individually and collectively to distinguish any changes in the amylase activity. As amylase itself could not be measured, the products of it were considered instead. As amylase converts starch to maltose, maltose concentrations were deliberated.

METHOD

The following concentrations were used, which contained a piece of exocrine pancreas, for detection of amylase/maltose production. The succeeding HEPES buffers were used, 1) with 1mM EGTA added (which was for the calcium free solution), 2) with no added EGTA (where additional calcium was able to be added).

Calcium

cAMP

CCh

cAMP + CCh

Without Calcium

1

1ml EGTA

0

2

0.5ml EGTA

0.5ml cAMP

3

3. 1ml EGTA

2μl CCh

4

0.5ml EGTA

0.5ml cAMP

2μl CCh

Buffer + Calcium

5

1ml Buffer

1mM

2μl CaCl₂

6

0.5ml buffer

0.5ml cAMP

2μl CaCl₂

7

1ml Buffer

2μl CaCl₂

2μl CCh

8

0.5ml Buffer

0.5ml cAMP

2μl CCh

2μl CaCl₂

Buffer + Calcium

9

1ml Buffer

4μM

8μl CaCl₂

10

0.5ml Buffer

0.5ml cAMP

8μl CaCl₂

11

0.5ml Buffer

2μl CCh

8μl CaCl₂

12

0.5ml buffer

0.5ml cAMP

2μl CCh

8μl CaCl₂FIGURE 1: shows the concentrations and amounts of agonist and buffer used for each solution.

AMYLASE

Ten times the diluted enzyme was put into a tube using x10 and x40 dilutions; this was then followed by the addition of 100µl of substrate which was left to stand for fifteen minutes. To stop the reaction 200µl of stopping reagent was added to the solution. This was then boiled for five minutes which was then left to cool before use. Subsequently, 2ml of distilled water was added. 190µl of the solution was transferred into each well of a 96 well plate the absorbance was measured at 540nm.

STANDARDS

The standards were made up using a stock solution of 50 µmoles maltose/ml

0, 0.025, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 4, 5 and 10

These standards were put in that the same time as the amylase assay so that both measurements were taken at the same time at 540nm absorbance.

RESULTS

Calcium

Abs at 540nm

Tube

cAMP

Abs at 540nm

Tube

CCh

Abs at 540nm

Tube

CCh & cAMP

Abs at 540nm

Tube

EGTA Buffer x 10

0.028

1

0.071

2

0.059

3

0.121

4

EGTA Buffer x 40

0.009

0.01

0.026

0.011

Buffer + 1mM Calcium x 10

0.055

5

0.065

6

0.042

7

0.058

8

Buffer + 1mM Calcium x 40

0.033

0.027

0.024

0.016

Buffer + 4µM Calcium x 10

0.056

9

0.098

10

0.072

11

0.147

12

Buffer + 4µM Calcium x 40

0.021

0.062

0.022

0.046 The following results illustrate the interaction of the acinar cells with no calcium (EGTA buffer), with calcium (1mM calcium present) and with calcium (4µM calcium present) in the presence of calcium, cAMP, Carbachol or CCh and cAMP together. They demonstrate the amylase activity by looking at the measure of maltose produced during these individual reactions in the form to radioactivity. This is then followed by the weight of pancreas present within each of the tubes which is later corrected for wet weight to 100mg/hr. Thereafter standards are produced with their absorbance at 540nm. Finally a graph to illustrate the amount of maltose produced.

TABLE 1: shows a measure of maltose produced in conjunction with calcium, cAMP, CCh or CCh and cAMP together. From looking at the results its can be seen that the two different buffers of calcium did not have much effect, as similar amounts of maltose was produced for both of the x10 concentrations and also the x40 concentration. Abs 540nm = absorbance at 540nm.

TABLE 2: Shows the weight of pancreas within each test tube which corresponds to TABLE 1.

Tube

Weight (mg)

1

18.6

2

10

3

9.4

4

25.1

5

18.7

6

15.8

7

5.3

8

8.3

9

12.8

10

8.3

11

11.2

12

9.4

TABLE 3: Demonstrates the absorbance at 540nm for each standard which is plotted as a graph in FIGURE 2. The absorbance increases as the concentration of the standards also increase.

STANDARD (µmoles)

ABSORBANCE AT 540nm

0

0.007

0.025

0.011

0.05

0.006

0.1

0.013

0.2

0.008

0.4

0.012

0.6

0.021

0.8

0.021

1

0.025

2

0.068

4

0.161

5

0.201

10

0.409

TABLE 4: The x10 concentration was incorporated and the values from TABLE 1 were marked off Figure 2 which provided the amount of maltose produced. The numbers below represent the value marked off FIGURE 2 multiplied by ten as this concentration was used instead of forty.

CALCIUM (mM)

Camp

(mM)

CCh

(mM)

CCh & cAMP (mM)

EGTA Buffer x 10

11.5

24.5

21.0

33.0

Buffer + 1mM Calcium x 10

20.0

22.5

16.0

20.8

Buffer + 4µM Calcium x 10

22.0

29.0

24.6

37.5

TABLE 5: This table gives the final concentrations of maltose which were obtained from FIGURE 2. The calculations for these values are shown in APPENDIX 1 and were corrected for 100mg/hr wet weight.

Calcium

(µm/100mg wet weight/ hr)

cAMP

(µm/100mg wet weight/ hr)

CCh

(µm/100mg wet weight/ hr)

cAMP & CCh

(µm/100mg wet weight/ hr)

EGTA Buffer

0.0185

0.0735

0.0670

0.0394

Buffer + 1mM Calcium

0.0321

0.0427

0.0906

0.0752

Buffer + 4μM Calcium

0.0795

0.105

0.0659

0.1197

FIGURE 2: Represents the values in TABLE 3 incorporated with the values in TABLE 4. The dependence of absorbance at 540nm is plot against the maltose concentrations.

DISCUSSION

The aim of this experiment was to observe the role of calcium in the secretion of amylase from the pancreas. From the results attained, it can be said that the x 10 concentration measured a higher amount of maltose compared to the x 40 concentration. Also, the buffers in conjunction with carbachol and with cAMP seemed to have produced an increased amount of maltose, in addition when they were measured together maltose concentration was increased further. This showed that using agonist's cAMP and carbachol; increased the amylase activity more than it did when calcium was used.

As cAMP produced elevated results it could be proposed that an intracellular signally pathway may have been involved. This may have been a measure through the release of intracellular stores of calcium from the endoplasmic reticulum, which can be stimulated via adenylyl cyclase or phospholipase C. If this was the case, the activation of adenylyl cyclase via a GPCR may have led to increased levels of cAMP thus releasing calcium via protein kinase A.

CCK-8 which is a hormone may have activated the enzyme phospholipase C through the muscuranic receptors found on the acinar cells which was stated by Metz et al, (1992). This could have led to the stimulation of converting PIP₂ into IP₃ and DAG. IP₃ is one the most common compounds that release intracellular stores of calcium. This may have occurred by unfolding them through the ryanodine receptors that are found within the endoplasmic reticulum (Galiano et al. 2003).

A number of studies have been carried out in the aim to show the release of amylase from pancreatic acinar cells in regards to intracellular calcium. O'Sullivan and Jamieson (1992) found that cAMP played a role in the secretion of amylase from pancreatic acinar cells of rats. O'Sullivan and Jamieson mention that another study suggested that the protein kinase A (PKA) involved with cAMP was the regulatory subunit instead of the catalytic and that in the presence of a protein kinase A inhibitor there was no reaction in the release of amylase however cAMP dependent phosphorylation was inhibited. This indication could support the results of the present experiment. Furthermore, they go on to state that PKA activation has an effect on amylase secretion whereas PKC activation does not have any effect on the activity of amylase. Therefore it may need to be considered whether the phospholipase C pathway is inconsistent with this data and the present data. A similar aspect is considered in the study of Metz et al (1992), they discovered that the enzyme bombesin which causes the secretion of amylase also increases intracellular calcium in acinar cells but it did not alter the secretion of amylase.

However, Metz et al (1992) carried out an investigation with the presence of thapsigargin which is a microsomal calcium ATPase inhibitor, to see whether it promoted a change in cellular calcium. They came to the conclusion that thapsigargin was involved in the release of intracellular stores of calcium via IP₃ dependent calcium release. From this they proposed that the thapsigargin prevented the refilling of the calcium stores in the endoplasmic reticulum, which elevated the levels of intracellular calcium and emptying the calcium stores so that the calcium was free in the cytoplasm of the cell. Metz et al (1992) believe that thapsigargin had temporarily increased the intracellular calcium in the acinar cells. They found that this temporary increase of calcium, decreased after sometime. This decrease occurred due to calcium influx; however the system is unknown for it. This is the reason for which extracellular calcium is important, it was stated that this hypothesis was agreed with in previous studies.

Stored calcium needs to be within a certain level so that it can sustain the secretion of enzymes in this case maltose. This was carried out by agonists that proceeded to increase IP₃ levels via PLC, which would in the end lead to excessive calcium which may need to be endorsed by rotation of cytoplasmic calcium. Metz et al (1992) believe that the rotation procedure may be disturbed by thapsisgargin as it would inhibit the calcium ATPase which would leave the calcium levels too low for stimulating the secretion of enzymes. This reaction was demonstrated for cAMP and carbachol however, in the presence of a calcium buffer and a calcium agonist the maltose concentration was relatively low. This could then be put forward as, adding too much calcium may reverse the effect of it and produce a reduced amount of maltose.

Argent et al (1982) suggested that the intracellular stores that distributed calcium in the cytoplasm is what triggered the reaction of secreting the pancreatic enzyme amylase rather than extracellular calcium. They also go on to suggest that different stimuli may deplete different parts of the endoplasmic reticulum for intracellular calcium. This suggests that the results obtained in the present study may be factual because cAMP and carbachol are both agonists of the muscuranic and acetylcholine receptors. This may have stimulated these receptors to activate intracellular signalling pathways leading to an increased amylase activity.

CONCLUSION

The results conclude that there is a high chance that intracellular signalling pathways are involved in the increase of amylase activity which was observed through cAMP and carbachol. It is probable that the phospholipase C and adenylyl cyclase are involved in this course of action as they are the two main pathways which lead to an increase in intracellular calcium via IP₃, protein kinase C and also protein kinase A. It could also be proposed that using calcium in the presence of a calcium buffer may overturn the amylase activity due to overwhelming of the system.

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