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Biomedical subjects

J B Hoek

Publications and source records attributed to J B Hoek.

At least 37 records · Page 2Linked to original sources
PubMed1998

Metabolic design: how to engineer a living cell to desired metabolite concentrations and fluxes.

A biotechnological aim of genetic engineering is to increase the intracellular concentration or secretion of valuable compounds, while making the other concentrations and fluxes optimal for viability and productivity. Efforts to accomplish this based on over-expression of the enzyme, catalyzing the so-called "rate-limiting step," have not been successful. Here we develop a method to determine the enzyme concentrations that are required to achieve such an aim. This method is called Metabolic Design Analysis and is based on the perturbation method and the modular ("top-down") approach-formalisms that were first developed for the analysis of biochemical regulation such as, Metabolic Control Analysis. Contrary to earlier methods, the desired alterations of cellular metabolism need not be small or confined to a single metabolite or flux. The limits to the alterations of fluxes and metabolite concentrations are identified. To employ Metabolic Design Analysis, only limited kinetic information concerning the pathway enzymes is needed.

Biotechnology↗
PubMed1998

Carbachol-stimulated Ca2+ increase in single neuroblastoma SH-SY5Y cells: effects of ethanol.

The effect of ethanol on the characteristics of carbachol-stimulated release of Ca2+ from intracellular Ca2+ stores was studied in single SH-SY5Y cells. Stimulation with carbachol, in the absence of extracellular Ca2+, elicited a rapid Ca2+ increase in SH-SY5Y cells peaking within seconds after addition of maximal agonist concentration. The Ca2+ response pattern in single cells resembled the population response, and there was no evidence of oscillatory changes in cytosolic [Ca2+] ([Ca2+]i). However, cell-to-cell variability could be detected in the magnitude and the latency time of the response, and in the rate of [Ca2+]i increase. In a carbachol dose-response analysis, the EC50 for the number of responsive cells and for the peak [Ca2+]i response was lower than that for carbachol-induced inositol 1,4,5-trisphosphate formation by a factor of 5 to 50. Ethanol (100 mM) caused a significant suppression of the number of responsive cells, but only when cells were stimulated with nonsaturating carbachol concentrations (1 and 10 microM). The suppression by ethanol was evident primarily in those cells that gave a Ca2+ response after several seconds of stimulation, whereas cells that responded within the initial seconds of receptor stimulation remained relatively unaffected. In responding cells stimulated with 10 microM carbachol, ethanol exposure also suppressed the maximal Ca2+ increase primarily in those cells that responded late. We suggest that ethanol suppression of muscarinic receptor-mediated signal transduction through the phospholipase C pathway may depend on the potentiation of feedback inhibition that requires receptor stimulation.

Calcium↗
PubMed1997

Quantification of information transfer via cellular signal transduction pathways.

A conceptual framework is developed for the quantitative analysis of signal transfer through cellular signal transduction pathways and networks. This approach is referred to as signal transfer analysis and is based on formalisms that were first developed for the analysis of metabolic networks. Signal transduction is quantified as the sensitivity, known as the response coefficient of a target (e.g. an ion channel or transcription factor) to a signal (e.g. a hormone, growth factor or neurotransmitter). This response coefficient is defined in terms of the fractional change in the activated target brought about by a small fractional change in the signal. Quantifying the signal transduction in this way makes it possible to prove that for an idealized signaling cascade without feedback loops, the total response equals the product of all the local response coefficients, one for each level of the cascade. We show under which conditions merely having more levels in a cascade can boost the sensitivity of a target to a signal. If a signal propagates to a target through two different routes, these routes contribute independently to the total response, provided there is no feedback from the target. This independence makes the behavior of signaling cascades different from that of metabolic pathways, where different branches are connected through Kirchhoffs law. The relations between the total response and the local kinetics at each level are given for a number of network structures, such as branched signaling pathways and pathways with feedback. The formalism introduced here may provide a general approach to quantify cellular information transfer.

Cell Physiological Phenomena↗
PubMed1997

Increased oxidative damage to mitochondrial DNA following chronic ethanol consumption.

Ethanol consumption adversely affects the structural and functional integrity of hepatic mitochondria. Some of these effects may arise from the increased cellular levels of oxidizing radicals induced by ethanol feeding. Since DNA is a potential target of free radical damage, we investigated the effect of chronic ethanol feeding on oxidative modification of mtDNA. Mitochondria were isolated from the livers of control and ethanol-fed rats and the mtDNA analyzed for the presence of 8-hydroxydeoxyguanosine (8-OHdG). A 21% increase in the level of 8-OHdG was detected in mtDNA from animals that had been fed an ethanol-containing diet for 42-76 days (control, 3.98 +/- 0.5; ethanol, 4.8 +/- 0.9 8-OHdG/100,000dG). This difference increased to 43% in animals that had been fed ethanol for 105-164 days (control, 6.9 +/- 1.0; ethanol, 9.9 +/- 1.1 8-OHdG/100,000dG). This increase in mtDNA oxidation was accompanied by a decrease in the recovery of mtDNA (42-76 days: control, 88 +/- 11; ethanol 76 +/- 8 ng/mg mitochondrial protein; 105-164 days: control, 88 +/- 13; ethanol 62 +/- 14 ng/mg mitochondrial protein). The data presented demonstrate that chronic ethanol feeding leads to increased oxidative damage of hepatic mtDNA. This may in turn result in progressive mtDNA mutations and deletions and impaired mitochondrial function.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1997

Packing constraints and electrostatic surface potentials determine transmembrane asymmetry of phosphatidylethanol.

The energetic determinants of the distribution of anionic phospholipids across a phosphatidylcholine (PtdCho) bilayer with different packing constraints in the two leaflets were studied, using (13)CH2-ethyl-labeled phosphatidylethanol (PtdEth) as a (13)C NMR membrane probe. PtdEth is unique in exhibiting a split (13)CH2-ethyl resonance in sonicated vesicles, the two components originating from the inner and outer leaflets, thus permitting the determination of the PtdEth concentration in each leaflet. Small and large unilamellar PtdEth-PtdCho vesicles were prepared in solutions of different ionic strengths. A quantitative expression for the transbilayer distribution of PtdEth, based on the balance between steric and electrostatic factors, was derived. The transbilayer difference in packing constraints was obtained from the magnitude of the PtdEth signal splitting. The electrostatic contribution could be satisfactorily described by the transmembrane difference in Gouy-Chapman surface potentials. At low (0.1-0.25%) PtdEth levels and high (up to 500 mM) salt concentrations, PtdEth had a marked fivefold preference for the inner leaflet, presumably because of its small headgroup, which favors tighter packing. At higher PtdEth content (4.8-9.1%) and low salt concentrations, where electrostatic repulsion becomes a dominant factor, the asymmetry was markedly reduced and an almost even distribution across the bilayer was obtained. In less curved, large vesicles, where packing constraints in the two leaflets are approximately the same, the PtdEth distribution was almost symmetrical. This study is the first quantitative analysis of the balance between steric and electrostatic factors that determines the equilibrium transbilayer distribution of charged membrane constituents.

Biophysical Phenomena↗
PubMed1997

Differential inhibition of epidermal growth factor signaling pathways in rat hepatocytes by long-term ethanol treatment.

BACKGROUND & AIMS: Long-term ethanol intake suppresses liver regeneration in vivo and ethanol interferes with epidermal growth factor (EGF)-induced DNA synthesis in vitro. Therefore, the effects of long-term ethanol treatment on EGF-activated signaling reactions in rat hepatocytes were investigated. METHODS: Hepatocytes from long-term ethanol-fed rats and pair-fed controls were stimulated with EGF (0.5-20 nmol/L) for 15-120 seconds. Tyrosine phosphorylation of EGF receptor (EGFR), Shc, and phospholipase-C gamma1 (PLC gamma), and growth factor receptor binding protein 2 (Grb2) coprecipitation with EGFR and Shc were analyzed by Western blotting. RESULTS: EGFR autophosphorylation was suppressed at all EGF concentrations in ethanol-fed cells compared with pair-fed cells, without significant differences in total EGFR protein or EGFR tyrosine kinase activity detected in cell lysates, suggesting that intracellular factors suppressed EGFR function. EGF-induced PLC gamma tyrosine phosphorylation and inositol 1,4,5-trisphosphate (InsP3) formation were suppressed, but cytosolic [Ca2+]c elevation was little affected, indicating enhanced InsP3-mediated intracellular Ca2+ release in ethanol-fed cells. Grb2 binding to EGFR was suppressed, but EGF-induced Shc tyrosine phosphorylation and Grb2 association with Shc were not significantly decreased. CONCLUSIONS: Long-term ethanol feeding suppressed EGF-induced receptor autophosphorylation in rat hepatocytes with differential inhibition of downstream signaling processes mediated by PLC gamma, Shc, and Grb2. Altered patterns of downstream signals emanating from EGFR may contribute to deficient liver regeneration in chronic alcoholism.

Animals↗
PubMed1997

Ethanol consumption and susceptibility of the pancreas to cerulein-induced pancreatitis.

Despite the fact that alcoholism is one of the major causes of pancreatitis, the pathogenesis of this disorder remains obscure. Factors such as the pattern of ethanol consumption, diet, and genetic predisposition may be contributing factors. The failure to produce alcoholic pancreatitis in experimental animals suggests that experimental provision of ethanol may only increase the predisposition to pancreatitis. To test this possibility, we developed an assay system using the in vitro model of cerulein-induced pancreatitis. In this system, pancreatic lobules were first exposed to a supraphysiologic concentration (10(-6) M) of the cholecystokinin analogue, cerulein, after which homogenates were incubated for up to 6 h. Activation of trypsinogen and chymotrypsinogen was observed only in cerulein-treated preparations. We then investigated the effects of the duration of ethanol feeding on cerulein-induced changes in rat pancreas. The pancreata from rats fed ethanol for 9-12 months were more susceptible to cerulein-induced activation of chymotrypsinogen compared to the pancreata from pair-fed control animals. This susceptibility also paralleled morphologic changes, such as dilatation of endoplasmic reticulum, only in the ethanol-fed group. In contrast, during the early stages (up to 3 months) of ethanol consumption, there was resistance (p < 0.01) to cerulein-induced changes. These results suggest that long-term ethanol consumption increases susceptibility to pancreatitis and raises the possibility that a similar mechanism may operate in human alcoholics.

Alcoholism↗
PubMed1997

Two mechanisms by which ATP depletion potentiates induction of the mitochondrial permeability transition.

The present and a previous study [J. W. Snyder, J. G. Pastorino, A. M. Attie, and J. L. Farber, Am. J. Physiol. 264 (Cell Physiol. 33): C709-C714, 1993] define two mechanisms whereby ATP depletion promotes liver cell death. ATP depletion and cell death are linked by the mitochondrial permeability transition (MPT). Mitochondrial deenergization promotes the MPT, and ATP maintains a membrane potential by reversal of ATP synthase. With an increased influx of Ca2+ induced by the ionophore A-23187, oligomycin depleted the cells of ATP without loss of the mitochondrial membrane potential and further elevated the intracellular Ca2+ concentration. Cyclosporin A (CyA) prevented the accompanying cell killing. Fructose also preserved the viability of the cells. With the increased cytosolic Ca2+ imposed by A-23187, viability is maintained by ATP-dependent processes. Upon depletion of ATP, Ca2+ homeostasis cannot be maintained, and the MPT is induced. Rotenone also depleted the cells of ATP, and A-23187 accelerated the loss of the mitochondrial membrane potential occurring with rotenone alone. CyA and fructose prevented the cell killing with rotenone and A-23187. Oligomycin did not prevent this action of fructose. We conclude that ATP is needed to maintain Ca2+ homeostasis to prevent the MPT and the resultant liver cell death. ATP is also needed to maintain mitochondrial energization when electron transport is inhibited.

Adenosine Triphosphate↗
PubMed1997

Hormonal stimulation, mitochondrial Ca2+ accumulation, and the control of the mitochondrial permeability transition in intact hepatocytes.

Ca2+ functions as an intracellular signal to transfer hormonal messages to different cellular compartments, including mitochondria, where it activates intramitochondrial Ca2(+)-dependent enzymes. However, excessive mitochondrial Ca2+ uptake can promote the mitochondrial permeability transition (MPT), a process known to be associated with cell injury. The factors controlling mitochondrial Ca2+ uptake and release in intact cells are poorly understood. In this paper, we investigate mitochondrial Ca2+ accumulation in intact hepatocytes in response to the elevation of cytosolic Ca2+ levels ([Ca2+]c) induced either by a hormonal stimulus (vasopressin), or by thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+ pump. After stimulation, cells were rapidly permeabilized for the determination of the mitochondrial Ca2+ content (Ca2+(m)) and to analyze the susceptibility of the mitochondria to undergo the MPT. Despite very similar levels of [Ca2+]c elevation, vasopressin and thapsigargin had markedly different effects on mitochondrial Ca2+ accumulation. Vasopressin caused a rapid (< 90 sec), but modest (< 2 fold) increase in Ca2+(m) that was not further increased during prolonged incubations, despite a sustained [Ca2+]c elevation. By contrast, thapsigargin induced a net Ca2+ accumulation in mitochondria that continued for up to 30 min and reached Ca2+(m) levels 10-20 fold over basal. Accumulation of mitochondrial Ca2+ was accompanied by a markedly increased susceptibility to undergo the MPT. Both mitochondrial Ca2+ accumulation and MPT activation were modulated by treatment of the cells with inhibitors of protein kinases and phosphatases. The results indicate that net mitochondrial Ca2+ uptake in response to hormonal stimulation is regulated by processes that depend on protein kinase activation. These controls are inoperative when the cytosol is flooded by Ca2+ through artificial means, enabling mitochondria to function as a Ca2+ sink under these conditions.

Animals↗
PubMed1996

Phosphatidylethanol as a 13C-NMR probe for reporting packing constraints in phospholipid membranes.

13CH2-ethyl labeled phosphatidylethanol (PEth), a rare naturally occurring anionic phospholipid, was used to probe the interleaflet packing density difference in small and large unilamellar phospholipid vesicles (SUVs and LUVs, respectively). The intrinsically tighter lipid packing in the inner leaflet of the SUVs resulted in the splitting of the CH2-ethyl 13C-resonance into two distinct components originating from PEth molecules residing in the inner and outer leaflets. The splitting of the 13C-NMR signal from the PEth headgroup appears to be unique among naturally occurring phospholipids. We present data suggesting that the splitting of the PEth signal reports on transleaflet packing density difference modulated by unequal electrostatic interactions and structured water on the inner and outer surfaces of the SUV. The PEth resonance splitting was insensitive to pH changes over the range 5.3-8.6 and cannot be accounted for by differences in the pKa of PEth in the inner and outer monolayers of the SUV. In 13C-NMR spectra of LUVs, where packing constraints in both monolayers are approximately similar, only a single, narrow symmetrical CH2-ethyl signal was observed, which was shifted downfield at higher PEth concentrations. The carbonyl and C3-glycerol backbone PEth resonances were shifted upfield compared to those of phosphatidylcholine or phosphatidylglycerol, suggesting a more tightly packed/hydrophobic environment for these segments of the PEth molecule in the membrane. We conclude that the unique splitting of the PEth 13C-resonance reported here can be used to characterize the lipid packing conditions in various membranes and to monitor the transbilayer distribution/movement of PEth.

Carbon Isotopes↗
PubMed1996

Ethanol-induced inhibition of cell proliferation is modulated by insulin-like growth factor-I receptor levels.

Ethanol inhibits the tyrosine autophosphorylation of the insulin-like growth factor (IGF)-I receptor, an action that correlates with the inhibition of IGF-I-stimulated cell proliferation [J. Biol. Chem. 268:21777-21782 (1993)]. In the current study, the IGF-I-dependent proliferation of mouse BALB/c3T3 cells was completely inhibited by ethanol, but the growth of BALB/c3T3 cells that overexpress the IGF-I receptor (p6 cells) was only partially inhibited by ethanol BALB/ c3T3 cells that simultaneously overexpress both the IGF-I receptor and IGF-I were insensitive to growth inhibition by ethanol. In p6 cells, increasing concentrations of IGF-I overcame the inhibition of IGF-I receptor tyrosine autophosphorylation in the presence of ethanol. The importance of the IGF-I receptor as a specific target for ethanol was further investigated in C6 rat giloblastoma cells that respond mitogenically to both epidermal growth factor (EGF) and IGF-I. The mitogenic response of C6 cells to EGF was abrogated in cells expressing antisense mRNA to the IGF-I receptor. Thus, EGF action in these cells is dependent on activation of an IGF-I/IGF-I receptor autocrine pathway. Indeed, EGF stimulated an increase in IGF-I receptor levels by more than 100%. Ethanol completely inhibited the proliferation of C6 cells in response to either EGF or IGF-I. However, ethanol did not directly interfere with EGF receptor function, because EGF-induced cell proliferation was unaffected by ethanol when added exclusively during a 1-hr exposure to EGF. Ethanol did not interfere with the EGF-induced increase in IGF-I receptor expression. The addition of both EGF and IGF-I overcame the inhibitory action of ethanol. In conclusion, the potency of ethanol as an inhibitor of IGF-I-mediated cell proliferation correlates with the level of IGF-I receptors. In contrast to its effect on the IGF-I-receptor, ethanol has no direct effect on EGF receptor activation.

Animals↗
PubMed1996

Interaction of protein phosphatases and ethanol on phospholipase C-mediated intracellular signal transduction processes in rat hepatocytes: role of protein kinase A.

Phospholipase C (PLC)-mediated signal transduction processes in rat hepatocytes are subject to modulation by protein phosphatases (PPases) and protein kinases, including protein kinase A (PKA) and protein kinase C. Ethanol (EtOH) stimulates PLC activity in liver cells in the absence of hormones, and EtOH pretreatment inhibits the subsequent stimulation of PLC by hormonal stimuli. There is evidence that protein kinase activities are involved in these actions of EtOH. We investigated the effects of okadaic acid (OKA), a PPase inhibitor, and 8-(4-chlorophenylthio)adenosine 3':5'-cyclic monophosphate (cpt-cAMP), a cell permeant cAMP analog that activates PKA, on EtOH-induced PLC activation. In addition, we studied the combined effects of cpt-cAMP and EtOH/OKA on vasopressin-induced PLC activation. PLC activation (cytosolic Ca2+ mobilization and inositol trisphosphate accumulation) induced by EtOH and vasopressin was inhibited by treatment with OKA, and was potentiated by cpt-cAMP. OKA treatment prevented the effect of cpt-cAMP. Pretreatment with EtOH caused inhibition of vasopressin-induced PLC activation. EtOH also decreased the enhancing effect of cpt-cAMP on the responses to vasopressin. The susceptibility to enhancement by cpt-cAMP plotted as a function of the initial rate of vasopressin-induced Ca2+ mobilization in EtOH-treated cells was similar to the pattern observed in OKA-treated cells. These data suggest that interactions of OKA and PKA on EtOH-induced PLC activation occurred at the level of G-protein, and indicate that EtOH may act as an inhibitory agent of PPase.

Animals↗
PubMed1996

Effect of glutathione on inositol 1,4,5-triphosphate-induced Ca2+ release in permeabilized hepatocytes from control and chronic ethanol-fed rats.

The effect of oxidized and reduced glutathione on inositol 1,4,5-trisphosphate (InsP3)-induced Ca2+ release from endoplasmic reticular Ca2+ stores was studied in digitonin-permeabilized hepatocytes from chronically ethanol-fed rats and pair-fed control animals. The fractional Ca2+ release induced by a subsaturating concentration of InsP3 was significantly enhanced in cells from ethanol-fed rats in the absence of a change in maximal InsP3-releasable Ca2+ pool size, and this difference was not affected by preincubation with reduced glutathione. Incubation with oxidized glutathione (1 mM) increased the efficacy of Ca2+ release by subsaturating concentrations of InsP3 in both control preparations and in cells from ethanol-fed rats. The shift in the InsP3 dose-response curve was not significantly different between the two preparations. These findings suggest that the enhanced efficacy of InsP3-induced Ca2+ release in hepatocytes from ethanol-fed rats is not caused by the oxidation of protein-bound thiol groups on the InsP3 receptor.

Alcoholism↗
PubMed1996

Inhibitory effect of ethanol on hepatocyte growth factor-induced DNA synthesis and Ca2+ mobilization in rat hepatocytes.

Hepatocyte growth factor (HGF) is the most potent mitogen identified for hepatocytes and is thought to be an important growth factor in the regulation of liver regeneration. Its effects are mediated through a tyrosine kinase receptor, the product of c-met proto-oncogene. One of the downstream signaling processes activated by HGF is phospholipase C-gamma. HGF stimulation of liver cells causes formation of inositol 1,4,5-triphosphate, which releases Ca2+ from intracellular Ca2+ ([Ca2+]i) stores, and causes elevation of cytosolic Ca2+ levels. It is known that liver regeneration is inhibited by both acute and chronic ethanol (EtOH) treatment. We investigated the effect of EtOH on HGF-induced DNA synthesis and mobilization of [Ca2+]i in rat hepatocytes in primary culture. DNA synthesis was monitored by [3H]thymidine incorporation in primary cultures of hepatocytes 42 hr after stimulation with HGF. HGF concentration required for maximum DNA synthesis was 0.3 to 1 ng/ml, and DNA synthesis was inhibited by 100 mM EtOH at HGF concentrations in the range of 0.1 to 5 ng/ml. This inhibition was strongest (45 to 47% inhibition) at a low concentration of HGF (0.1 to 0.3 ng/ml) and decreased at an HGF concentration > 1 ng/ml. HGF-induced changes in [Ca2+]i were measured in single fura 2-loaded hepatocytes by fluorescence imaging techniques. The Ca2+ response induced by HGF (0.3 to 5 ng/ml) was inhibited by EtOH, with an EC50 of approximately 50 mM. Analysis of Ca2+ response patterns in individual cells indicated that EtOH suppressed the number of responsive cells and made Ca2+ responses more transient, but did not affect peak [Ca2+]i elevation; thus suggesting an inhibition at the level of phospholipase C-gamma-activation. These data indicate that inhibition by EtOH of the response of liver cells to HGF may contribute to the inhibitory effect of EtOH on liver regeneration.

Animals↗
PubMed1995

Secretion of prostaglandins elicited by lipopolysaccharide and ethanol in cultured rat Kupffer cells.

Prostaglandins (PGs) released by cultured rat Kupffer cells in response to stimulation with lipopolysaccharide (LPS) or ethanol were extracted from culture media, separated by HPLC and measured by radioimmunoassay. LPS (0.5-5 micrograms/ml) enhanced, after a 3-4 hrs lag period, the production of PGE2 (7-10 fold by 24 hrs), thromboxane B2 (2-3 fold) and PGD2. PG 6-keto-F1 alpha, PGF2 alpha (20-50% each). This effect was not inhibited by 30 microM aspirin but was reduced by dexamethasone. Ethanol (25-85 mM) gradually increased the release of PGE2 (40-90% by 24 hrs) and other PGs (10-30%), with 30 microM aspirin eliminating this effect. When added together with LPS, ethanol potentiated the endotoxin action. We suggest that LPS causes synthesis of the inducible cyclooxygenase-2 form in Kupffer cells, whereas ethanol exerts its effect via the pre-existing cyclooxygenase-1 mainly by increasing the free arachidonic acid content.

Animals↗
PubMed1995

Ethanol inhibits the peak of muscarinic receptor-stimulated formation of inositol 1,4,5-trisphosphate in neuroblastoma SH-SY5Y cells.

The effect of ethanol on muscarinic receptor-stimulated formation of inositol 1,4,5-trisphosphate was studied in human neuroblastoma SH-SY5Y cells. Stimulation with carbachol induced a biphasic increase of inositol 1,4,5-triphosphate with an initial peak after 10 sec declining to a plateau phase of elevation above basal levels, which was sustained for at least 5 min in the presence of agonist. The peak, but not the plateau phase, was concentration-dependently decreased by exposure to ethanol. Maximal inhibition was obtained within 30 sec of exposure to ethanol. Ethanol caused an increase in the EC50 value of carbachol for the initial rate of inositol 1,4,5-trisphosphate formation, measured after 10 sec of stimulation, from 98 microM in the absence to 196 microM in the presence of 100 mM ethanol. The potencies of pirenzepine and hexahydro-sila-difenidol hydrochloride for inhibiting [3H]quinuclidinyl benzilate binding and inositol 1,4,5-trisphosphate formation suggest that both phases are mediated via the muscarinic M1 receptor. Phorbol 12-myristate 13-acetate inhibited both phases of inositol 1,4,5-trisphosphate formation, whereas okadaic acid and modulators of cAMP-dependent protein kinase were without any effect. There was no inhibitory effect of ethanol when protein kinase C was inhibited by H7 and calphostin C, indicating that the ethanol effect is dependent on protein kinase C activity.

Carbachol↗