Ravindra K. Malhotra

Exendin-4, a new peptide from Heloderma suspectum venom, potentiates cholecystokinin-induced amylase release from rat pancreatic acini.

We examined the actions of exendin-4, a new peptide isolated from Heloderma suspectum venom, on dispersed acini from rat pancreas. Exendin-4 caused a 3-fold increase in cAMP but did not alter cellular calcium concentration. Exendin-4-induced increases in cAMP were inhibited by an exendin-receptor antagonist, exendin (9-39)NH2, but not by VIP-receptor antagonists. Whereas up to 1 microM exendin-4 alone did not alter amylase release, potentiation of enzyme release was observed when the peptide (greater than 30 pM) was combined with cholecystokinin. Potentiation of amylase release was also observed when exendin-4 was combined with carbamylcholine, bombesin or a calcium ionophore, A23187. These results indicate that stimulation of exendin receptors on rat pancreatic acini causes an increase in cellular cAMP. Although this increase in cAMP alone does not result in amylase release, combination of exendin-4 with agents that increase cell calcium results in potentiation of amylase release.

Excess K+ and Phorbol Ester Activate Protein Kinase C and Support the Survival of Chick Sympathetic Neurons in Culture

Abstract: The effects of phorbol esters were investigated on the survival of chick sympathetic neurons in a serum‐free culture medium. The protein kinase C activator phorbol 12,13‐dibutyrate (PDB) supported about 40% of the plated sympathetic neurons. This number was comparable to that supported by nerve growth factor (NGF). A combination of phorbol ester and NGF did not significantly increase the number of surviving neurons. Phorbol ester‐supported sympathetic neurons possessed desipramine‐sensitive [3H]‐norepinephrine uptake mechanism, and therefore were noradrenergic in character. Two days after the start of cultures, if NGF was replaced by phorbol ester, or phorbol ester was replaced by NGF, the number of surviving sympathetic neurons was essentially the same in both groups, and the uptake of [3H]norepinephrine was also comparable when examined 2 days after the switchover. Interchangeability between phorbol ester and NGF in the survival of sympathetic neurons suggests that both agents act on the same subpopulation of neurons of the chick sympathetic ganglia. The protein kinase C activity of cytosol and particulate fractions of NGF‐supported neurons was 0.14 and 0.09 pmol/min/mg protein, respectively. In phorbol estersupported neurons the activity in the paniculate fraction increased by about fivefold. Removal of the phorbol ester after 2 days resulted in restoration of the enzyme activity in <1 h, and readdition of the phorbol ester again increased the activity by fivefold. When NGF was added to these neurons (1 μg for 15 min), there was no change in the enzyme activity. Phorbol 13‐acetate was ineffective in supporting sympathetic neurons in culture, as well as in enhancing protein kinase C activity. We also compared the protein kinase C activity of sympathetic neurons supported in culture by NGF and excess potassium (35 mM K+). Neurons supported in culture by 35 mM K+ for 2 days had almost eightfold more protein kinase C activity in their particulate fraction than in cytosol fraction. If NGF‐supported neurons were acutely treated with excess K+, the protein kinase C activity was increased in the particulate fraction by about sevenfold in a concentration‐ and time‐dependent manner. Excess K+ plus phorbol ester did not produce an additive effect on protein kinase C activity. PDB and excess K+ had no effect on cyclic AMP content of sympathetic neurons. In summary, the present data suggest that the neurotrophic action of PDB and excess K+ is probably mediated through protein kinase C.

PACAP-38, a novel peptide from ovine hypothalamus, is a potent modulator of amylase release from dispersed acini from rat pancreas

Despite studies indicating the presence of specific pancreatic acinar receptors for PACAP-38, a peptide that was recently isolated from ovine hypothalamus, the actions of the new peptide on pancreatic enzyme secretion have not been examined. The present study demonstrates that in terms of cAMP production and amylase release from dispersed acini from rat pancreatic acini, PACAP-38 and an N-terminal fragment, PACAP-27, have the same potency and efficacy as vasoactive intestinal peptide (VIP). As with VIP, these actions are potentiated by adding an inhibitor of cyclic nucleotide phosphodiesterase, and combination of PACAP-38 with bombesin, CCK-8, carbachol or the calcium ionophore A23187 results in 2-fold augmentation of the secretory actions of these agents. Inhibition of PACAP-38-induced cAMP production and amylase release by two VIP-receptor antagonists indicates that the secretory effects of PACAP-38 are mediated by interaction with VIP receptors. PACAP-38, a new brain-gut peptide, may be a physiological modulator of pancreatic enzyme secretion.

Comparison of secretion of catecholamines from the rat adrenal medulla during continuous exposure to nicotine, muscarine or excess K

The effects of continuous exposure of chromaffin cells to nicotine, muscarine and excess potassium on the secretion of catecholamines and uptake of radiolabeled calcium were investigated in the isolated perfused adrenal gland of the rat. Perfusion with 20 microM nicotine-Krebs solution increased catecholamine secretion from a basal value of 5 ng to 273 ng in the first 5 min. The secretion declined to about 20 ng after 30 min of continuous exposure to nicotine (slow desensitization). 200 microM muscarine increased the secretion to about 150 ng within the first 5-10 min, and remained at the level of 110 ng even after continuous perfusion with muscarine for up to 40 min (no desensitization). About 700 ng of catecholamines were secreted within the first 5 min of exposure to 55 mM K, the secretion declined to about 350 ng in the next 5 min, and remained almost at this level for 120 min during continuous perfusion with excess K (partial desensitization). When the secretion of catecholamines was depressed by continuous exposure to nicotine for 30 min, introduction of 55 mM K or 200 microM muscarine (along with nicotine) still led to a large increase in the secretion of catecholamines. Omission of calcium ions from the perfusion medium containing either nicotine or 55 mM K caused a rapid decline in the secretion of catecholamines, suggesting influx of external calcium, but muscarine-induced secretion was only modestly reduced by calcium removal, suggesting mobilization of internal calcium. Addition of EGTA to calcium-free medium completely abolished the secretion evoked by all agents.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein Kinase C of Sympathetic Neuronal Membrane Is Activated by Phorbol Ester-Correlation Between Transmitter Release, 45Ca2+ Uptake, and the Enzyme Activity

Abstract: The effects of phorbol esters [phorbol 12,13‐dibutyrate (PDB), 12‐O‐tetradecanoylphorbol 13‐acetate (TPA), and phorbol 13‐acetate] were investigated on the release of [3H]norepinephrine, 45Ca2+ accumulation, and protein kinase C activity in cultured sympathetic neurons of the chick embryo. Sympathetic neurons derived from 10‐day‐old chick embryo were cultured in serum‐free medium supplemented with insulin, transferrin, and nerve growth factor. After 3 days, neurons were loaded with [3H]‐norepinephrine and the release of [3H]norepinephrine was determined before and after electrical stimulation. Stimulation at 1 Hz for 15 s increased the release of [3H]‐norepinephrine over the nonstimulation period. Stimulation‐evoked release gradually declined with time during subsequent stimulation periods. Incubation of neurons in Ca2+‐free Krebs solution containing 1 mM EGTA completely blocked stimulation‐evoked release of [3H]‐norepinephrine. Stimulation‐evoked release of [3H]‐norepinephrine was markedly facilitated by 3 and 10 nM PDB or TPA. The spontaneous release was also enhanced by PDB and TPA. The net accumulation of 45Ca2+ during stimulation of sympathetic neurons was increased by two‐to fourfold in the presence of PDB or TPA. PDB at 1–100 nM produced a concentration‐dependent increase in the activation of protein kinase C. PDB at 30 nM increased the activity of protein kinase C of the paniculate fraction from 0.09 to 0.58 pmol/min/mg protein. There was no significant change in protein kinase C activity of the cytosolic fraction (0.14 pmol/min/mg versus 0.13 pmol/min/mg protein). The ratio of the paniculate to cytosolic protein kinase C increased from a control value of 0.62 to 4.39 after treatment with 30 nM PDB. TPA (10 and 30 nM) also increased protein kinase C activity of the paniculate fraction by six‐ to eightfold. Phorbol 13‐acetate had no effect on protein kinase C activity, [3H]norepinephrine release, and 45Ca2+ accumulation. These results provide direct evidence that activation of protein kinase C enhances Ca2+ accumulation, which in turn leads to the facilitation of transmitter release in sympathetic neurons.

Expression and phosphorylation of a MARCKS-like protein in gastric chief cells: further evidence for modulation of pepsinogen secretion by interaction of Ca2+/calmodulin with protein kinase C.

In gastric chief cells, agents that activate protein kinase C (PKC) stimulate pepsinogen secretion and phosphorylation of an acidic 72‐kDa protein. The isoelectric point and molecular mass of this protein are similar to those for a common PKC substrate; the MARCKS (for Myristoylated Alanine‐Rich C Kinase Substrate) protein. We examined expression and phosphorylation of the MARCKS‐like protein in a nearly homogeneous suspension of chief cells from guinea pig stomach. Western blotting of fractions from chief cell lysates with a specific MARCKS antibody resulted in staining of a myristoylated 72‐kDa protein (pp72), associated predominantly with the membrane fraction. Using permeabilized chief cells. we examined the effect of PKC activation (with the phorbol ester PMA), in the presence of basal (100 nM) or elevated cellular calcium (1 μM), on pepsinogen secretion and phosphorylation of the 72‐kDa MARCKS‐like protein. Secretion was increased 2.3‐, 2.6‐, and 4.5‐fold by incubation with 100 nM PMA, 1 μM calcium, and PMA plus calcium, respectively. A PKC inhibitor (1 μM CGP 41 251) abolished PMA‐induced secretion, but did not alter calcium‐induced secretion. This indicates that calcium‐induced secretion is independent of PKC activation. Chief cell proteins were labeled with 32P‐orthophosphate and phosphorylation of pp72 was detected by autoradiography of 2‐dimensional polyacrylamide gels. In the presence of basal calcium PMA (100 nM) caused a > two‐fold increase in phosphorylation of pp72. Without PMA, calcium did not alter phosphorylation of pp72. However, 1 μM calcium caused an approx. 50% attenuation of PMA‐induced phosphorylation of pp72. Experiments with a MARCKS “phosphorylation/calmodulin binding domain peptide” indicated that calcium/calmodulin inhibits phosphorylation of pp72 by binding to the phosphorylation/calmodulin binding domain and not by inhibiting PKC activity. These observations support the hypothesis that, in gastric chief cells, interplay between calcium/calmodulin binding and phosphorylation of a common domain on the 72‐kDa MARCKS‐like protein plays a role in modulating pepsinogen secretion. J. Cell. Biochem. 64:514–523.

McN-A-343, a specific agonist of M1-muscarinic receptors, exerts antinicotinic and antimuscarinic effects in the rat adrenal medulla.

Pirenzepine, McN-A-343 and oxotremorine were used to determine the subtypes of muscarinic receptors involved in the secretion of catecholamines from the isolated perfused adrenal gland of the rat. In the presence of 0.1 microM pirenzepine, the concentration-secretion curve for muscarine was shifted in parallel to the right by almost one log unit. With 0.5 microM the shift was over two log units. The apparent dissociation constant for pirenzepine was about 1.12 X 10(-8) M. Perfusion with McN-A-343 (1-30 microM) did not evoke the secretion of catecholamines. A further increase to very high concentrations (100-1000 microM) caused only a modest secretion (about 50 ng/5 min with 300 microM as compared to the same amount of secretion obtained with 1 microM muscarine). Secretion evoked by nicotine was significantly reduced (30%) by 3 microM McN-A-343, and the inhibition increased (90%) with higher concentrations (100 microM). McN-A-343 also produced concentration-dependent inhibition of catecholamine secretion evoked by muscarine. A significant effect was observed at 30 microM and reached a maximum level at 300 microM. Oxotremorine, like McN-A-343 was a partial agonist on the muscarinic receptors; but unlike McN-A-343, did not block the stimulatory effects of nicotine. Although the pirenzepine data suggest that M1 receptors are responsible for the secretion of catecholamines in the rat adrenal medulla, this conclusion is not supported by the results obtained with the M1-receptor agonist, McN-A-343, which proved to be an effective blocker of muscarinic as well as nicotinic receptors.

Regulation of calcium-induced exocytosis from gastric chief cells by protein phosphatase-2B (calcineurin)

The molecular mechanisms whereby calcium stimulates secretion are uncertain. In the present study, we used streptolysin O (SLO)-permeabilized chief cells from guinea pig stomach to investigate whether protein phosphatase-2B (calcineurin), a calcium/calmodulin-dependent, serine/threonine phosphatase plays a role in mediating calcium-induced pepsinogen secretion. Preincubation of cells with alpha-naphthylphosphate, a non-specific phosphatase inhibitor, decreased calcium-induced secretion. Likewise, specific inhibitors of protein phosphatase-2B (cyclosporin-A and FK-506) caused a dose-dependent reduction in calcium-induced pepsinogen secretion. Moreover, in intact cells, cyclosporin-A and FK-506 inhibited pepsinogen secretion caused by cholecystokinin, carbamylcholine and A23187, agonists known to increase chief cell cytosolic calcium. Okadaic acid, an inhibitor of protein phosphatase-1 and -2A, had no effect on secretion caused by these agonists. Chief cell calcium-dependent phosphatase activity, measured using radiolabeled casein as substrate, was reduced selectively by inhibitors of protein phosphatase-2B. Endogenous substrates for calcium/calmodulin-dependent phosphatase activity were identified by analyzing chief cell lysates using 2-dimensional gel electrophoresis. Increasing the cytosolic calcium concentration resulted in dephosphorylation of a 55-kDa, acidic cytoskeletal protein. FK-506 inhibited dephosphorylation of this protein. Thus, in permeabilized chief cells, specific inhibitors of protein phosphatase-2B inhibit calcium-induced pepsinogen secretion, calcium/calmodulin-dependent phosphatase activity and calcium-induced dephosphorylation of a 55-kDa, acidic cytoskeletal protein. These results support the hypothesis that protein phosphatase-2B (calcineurin) plays an important role in mediating calcium-induced exocytosis.

Formation of inositol trisphosphate by muscarinic agents does not stimulate transmitter release in cultured sympathetic neurons

Sympathetic neurons obtained from 10-day-old chick embryos were cultured and used to measure phospholipid hydrolysis and the release of [3H]norepinephrine ([3H]NE) after treatment with muscarinic agents. Acetylcholine (1-100 pM) produced large increases (200-1500%) in the formation of [3H]inositol tris ([3H]IP3), bis([3H]IP2) and mono ([3H]IP) phosphates. Muscarinic receptor agonist, muscarine, increased [3H]IP formtion by 600%. Atropine (10 nM) antagonized the effects of muscarine. Nicotine (3-100/~M) did not increase [3H]IP formation. Acetylcholine (10 and 100/~M) failed to enhance the release of tritium from [3H]NE-loaded sympathetic neurons, but addition of an excess of K produced a large increase. These data suggest that sympathetic neurons possess muscarinic receptors, which are coupled to phospholipid hydrolysis. Lack of [3H]NE release by acetylcholine indicates that sympathetic neurons do not contain a significant pool of bound Ca, which can be mobilized by [3H]IP3 generated by the action of acetylcholine. Increase in free cellular Ca is responsible for initiation of a neurosecretory response in many synapses. Concentration of free Ca within a cell is increased by either mobilization from intracellularly bound stores or from increasing the influx from the extracellular fluid. In the case of bovine chromaflin cells, it has been well documented that stimulation of nicotinic receptors causes an increase in Ca 45 uptake, leading to secretion of catecholamines [6, 9]. Muscarinic receptor activation produces an increase in [3H]IP3 levels in these chromaffin cells, but such an event is not associated with

Veratrine supports the in vitro survival of embryonic chick sympathetic neurons.

Veratrine (VT), an alkaloid known to act on the sodium channels and cause depolarization of a cell membrane, was found to support the survival of cultured sympathetic neurons. At 30 microM it was as effective as nerve growth factor (NGF), as determined by the cell counts and [3H]norepinephrine ([3H]NE) uptake. Protein kinase C (PKC) activity of the surviving neurons was measured because of our previous finding that depolarizing concentrations of K+ support the survival and cause several fold increase in the enzyme activity. An acute treatment of NGF-supported sympathetic neurons by VT did not alter PKC activity.