Mathematical modeling of skin papilloma data in SENCAR mice

High concentrations of pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors have been detected in the rat cerebellum during ontogenesis. In particular, PACAP receptors are actively expressed in immature granule cells, suggesting that PACAP may act as a neurotrophic factor in the developing rat cerebellum. In the present study, we have investigated the effect of PACAP on cell survival and neurite outgrowth in cultured immature cerebellar granule cells. In control conditions, cultured granule cells undergo programmed cell death. Exposure of cultured cells to PACAP for 24 and 48 h provoked a significant increase in the number of living cells. The effect of PACAP on cell survival was inhibited by the PACAP antagonist PACAP(6-38). Vasoactive intestinal polypeptide was approximately 1000 times less potent than PACAP in promoting cell survival. PACAP also induced a significant increase in the number of processes and in the cumulated length of neurites borne by cultured neuroblasts. The present results demonstrate that PACAP promotes cell survival and neurite outgrowth in cultured immature granule cells. Since PACAP and its receptors are expressed in situ in the rat cerebellar cortex, these data strongly suggest that PACAP plays a physiological role in the survival and differentiation of cerebellar granule cells.     

Expression of pituitary adenylate cyclase activating polypeptide (PACAP) in the postnatal and adult rat cerebellar cortex

In adult rat, immunostaining for pituitary adenylate cyclase activating polypeptide (PACAP) was demonstrated in the soma and dendrites of Purkinje cells and in nerve fibres around granule cells. PACAP in the Purkinje cells was confirmed by the presence of mRNA. The concentration of PACAP-38 was high after birth and declined to adult levels within a few weeks. At birth PACAP-immunoreactive nerve fibres and a few cells were present in the Purkinje cell layer, but already at postnatal day 7 an adult PACAP immunostaining pattern was found. The findings suggest that PACAP could be a neurotransmitter in the adult cerebellum and provide a morphological correlate for the reported in vitro anti-apoptotic PACAP effects on cerebellar granule cells from 1-week-old rats.     

Glucocorticoid receptor binding in proliferating and differentiating cells: modulation by a tumour promoting agent

The effects of the tumour-promoting agent, phorbol 12-myristate 13-acetate, on dexamethasone binding and protein kinase C in proliferating and differentiating murine erythroleukemic cells were investigated. Concentration dependent changes in steroid binding were observed in proliferating and differentiating cells treated with phorbol 12-myristate 13-acetate for 2h or 24h. Ligand binding in differentiating cells was consistently lower than in proliferating cells. Reduced expression of protein kinase C was observed in differentiating cells as compared with proliferating cells; the amount of enzyme protein was reduced on treatment of cells with phorbol ester.     

Differential signal transduction by five splice variants of the PACAP receptor

The two forms of pituitary adenylyl cyclase-activating polypeptide (PACAP-27 and -38) are neuropeptides of the secretin/glucagon/vasoactive intestinal polypeptide/growth-hormone-releasing hormone family and regulate hormone release from the pituitary and adrenal gland. They may also be involved in spermatogenesis, and PACAP-38 potently stimulates neuritogenesis and survival of cultured rat sympathetic neuroblast and promotes neurite outgrowth of PC-12 cells. The PACAP type-I receptor (found in hypothalamus, brain stem, pituitary, adrenal gland and testes), specific for PACAP, is positively coupled to adenylyl cyclase and phospholipase C. The recently cloned type II receptor does not discriminate between PACAP and vasoactive intestinal polypeptide and is coupled to only adenylyl cyclase. Here we have used a new expression cloning strategy, based on the induction of a reporter gene by cyclic AMP, to isolate a complementary DNA encoding the type-I PACAP receptor. On transfection of this cDNA, both PACAP-27 and -38 stimulate adenylyl cyclase with similar EC50 values (50% effective concentration, 0.1-0.4 nM), whereas only PACAP-38 stimulates phospholipase C with high potency (EC50 = 15 nM). Four other splice variants were isolated with insertions at the C-terminal end of the third intracellular loop. Expression of these cDNAs revealed altered patterns of adenylyl cyclase and phospholipase C stimulation, suggesting a novel mechanism for fine tuning of signal transduction.     

Chronic oxytocin pretreatment inhibits adenylyl cyclase activity in cultured rat myometrial cells

To determine whether chronic oxytocin pretreatment inhibits adenylyl cyclase, we compared adenylyl cyclase activity in membranes prepared from cultured, immortalized rat myometrial cells that were untreated or pretreated for 24 h with oxytocin. Chronic oxytocin pretreatment (1 x 10(-5) M for 24 h) attenuated basal, guanosine triphosphate (1 x 10(-5) M)-, isoproterenol (1 x 10(-4) M)-, forskolin (1 x 10(-5) M)-, MnCl2 (20 mM)- or NaF (1 x 10(-2) M)-stimulated adenylyl cyclase activity by 27 +/- 5% to 39 +/- 11% (n = 6, p < 0.05). Oxytocin pretreatment for 2 h (n = 5) did not produce a significant effect. To understand the mechanism by which oxytocin pretreatment decreased activity of the adenylyl cyclase pathway, we compared effects of pretreatment with either oxytocin or phenylephrine on adenylyl cyclase activity and determined the effects of Gi inhibition and protein kinase C (PKC) depletion. Chronic (24 h) phenylephrine pretreatment (1 x 10(-4) M) had effects similar to those of oxytocin pretreatment (1 x 10(-5) M). PKC depletion with phorbol 12-myristate 13-acetate (1 x 10(-6) M, 41 h) prevented attenuation of adenylyl cyclase activity by oxytocin pretreatment (1 x 10(-5) M for 24 h). Inhibition of Gi by pertussis toxin pretreatment (1.25 microg/ml, 41 h) had no significant effect. These findings suggest that chronic oxytocin pretreatment desensitizes the adenylyl cyclase pathway by a cross-regulatory mechanism that involves activation of Gq and PKC.     

Regulation of adenylyl cyclase by membrane potential

Mammalian adenylyl cyclases possess 12 transmembrane-spanning domains and bear a superficial resemblance to certain classes of ion channels. Some evidence suggests that bacterial and sea urchin sperm adenylyl cyclases can be regulated by membrane depolarization. In the present study, we explored the effect of altering membrane potential on the adenylyl cyclase activity of cerebellar granule cells with acute potassium depolarization. A biphasic stimulatory and then inhibitory response is evoked by progressive increases in the extracellular [K]:[Na] ratio in the absence of extracellular Ca2+. This effect does not mimic the linear increase in membrane potential elicited under the same conditions. Instead it appears as though membrane depolarization opens L-type (nimodipine-sensitive) Ca2+ channels, allowing the entry of Na+, which directly stimulates adenylyl cyclase activity. Gramicidin, which generates pores that are permeable to monovalent cations, and concurrently eliminates the membrane potential, permits a similar stimulation by extracellularly applied Na+. Although the results indicate no direct sensitivity of cerebellar granule cell adenylyl cyclase to membrane potential, they do demonstrate that, as a result of membrane depolarization, the influx of Na+, as well as Ca2+, will elevate cAMP levels.     

Gi-mediated stimulation of type II adenylyl cyclase is augmented by Gq-coupled receptor activation and phorbol ester treatment

Synergism between Gs- and Gi- or Gq-dependent signaling pathways has been demonstrated in the stimulation of type II adenylyl cyclase (AC-II). Provision of activated alpha s is known to allow numerous Gi-coupled receptors to stimulate AC-II and to potentiate the responses to Gq-coupled receptors. To explore possible interactions between Gi- and Gq-coupled receptors that are independent of alpha s, the activity of AC-II was determined after the activation of Gi- and Gq-regulated pathways. Human embryonic kidney 293 cells were transiently cotransfected with cDNAs encoding AC-II and various G-protein-coupled receptors. Agonist-bound Gi-coupled receptors (including the formyl peptide, dopamine-D2, and delta-opioid receptors) stimulated AC-II activity in the absence of activated alpha s, provided that the cells were treated with 100 nM phorbol 12-myristate 13-acetate. Activation of protein kinase C (PKC) thus appears to relieve the requirement for the presence of activated alpha s. Stimulation of PKC via Gq-coupled receptors also allowed Gi-coupled receptors to activate AC-II. Coexpression of the m1 muscarinic receptor with the dopamine-D2 receptor permitted dopamine to stimulate AC-II in the presence of carbachol. The phorbol ester-permissive and alpha s-independent stimulation was mediated by G-protein beta gamma subunits because it was blocked by the beta gamma scavengers alpha t and beta-adrenergic receptor kinase. These results show that AC-II can efficiently integrate signals generated by Gq- and Gi-coupled receptors via a mechanism that is independent of Gs.     

Changes in responsiveness of freshly isolated longitudinal muscle cells from rat uterus towards oxytocin during gestation: contractility and calcium signaling

The effect of ethanol exposure on muscarinic receptor-stimulated expression of c-fos was investigated in SH-SY5Y cells. Four days of ethanol exposure enhanced carbachol-stimulated c-fos mRNA expression, analyzed with Northern blot, and Fos/AP-1 binding activity, measured with gel mobility super shift assay. Pre-incubation with muscarinic antagonists or the protein kinase C inhibitor GF109203X demonstrated that, in both control and ethanol-treated cells, carbachol-induced c-fos expression was mediated via muscarinic M1 receptors and to a large extent through protein kinase C. However, phorbol ester-induced c-fos expression was unaffected in ethanol-treated cells. Acute exposure to ethanol caused a suppression of both carbachol- and phorbol ester-stimulated c-fos expression. These results demonstrate that muscarinic receptor-stimulated gene expression is sensitive to both acute and long-term ethanol exposure.     

Tuberculosis control in health care workers: an algorithmic approach

Smooth muscle cells isolated from cecal circular smooth muscle of the guinea pig were used to determine whether thyrotropin-releasing hormone (TRH) can inhibit the contractile response produced by 10(-6) M carbachol by exerting a direct action on muscle cells. In addition, the inhibitory effect of 2',5'-dideoxyadenosine (an inhibitor of adenylate cyclase), phorbol 12-myristate 13-acetate (an inhibitor of particulate guanylate cyclase), 6-anilinoquinoline-5,8-quinone (an inhibitor of nitric oxide synthase) on the TRH-induced relaxation of cecal circular smooth muscle cells was examined. TRH inhibited the contractile response produced by 10(-6) M carbachol in a concentration-dependent manner, with an IC50 value of 4 nM, 2',5'-Dideoxyadenosine and phorbol 12-myristate 13-acetate did not have any significant effect on the TRH-induced relaxation. On the other hand, 6-anilinoquinoline-5,8-quinone and N omega-nitro-L-arginine methyl ester significantly inhibited the relaxation produced by TRH. Our findings show that TRH has a direct inhibitory effect on the isolated cecal circular smooth muscle cells via activation of nitric oxide synthase and soluble guanylate cyclase.     

Change of ganglioside accessibility at the plasma membrane surface of cultured neurons, following protein kinase C activation

While the mechanism of signal transduction across the plasma membrane from the exo- to the endoplasmic side has been extensively investigated, the possible return of messages back to the outer layer is less known. We studied the effect of protein kinase C activation on the ganglioside accessibility at the exoplasmic face of intact rat cerebellar granule cells in culture, using the enzyme sialidase as the probing molecule. Under the experimental conditions (1 milliunit/mL enzyme, 2 min incubation at 37 degreesC), only GT1b and GD1a gangliosides were partially affected by the enzyme (28.6 and 25.7% hydrolysis, respectively). After cell treatment with phorbol 12-myristate 13-acetate, inducing protein kinase C activation, GT1b and GD1a ganglioside susceptibility to sialidase was strongly decreased (8.6 and 15.9% hydrolysis, respectively). A reduction of ganglioside hydrolysis was also observed when protein kinase C activation was induced by cell treatment for 15 min with 100 microM glutamate. On the contrary, accessibility did not vary when protein kinase C translocation was not effective (either in the absence of Ca2+ in the medium or using 1 microM glutamate) or when the kinase activity was inhibited by staurosporine. These data suggest that following PKC activation, a key step of inbound transmembrane signaling, cell may dispatch outbound messages to the plasma membrane outer layer, changing the selective recognition and crypticity of glycolipids at the cell surface, possibly through a modulation of their segregation state.     

Thrombin and phorbol esters potentiate Gs-mediated cAMP formation in intact human erythroid progenitors via two synergistic signaling pathways converging on adenylyl cyclase type VII

In intact, but not in permeabilized, human erythroid progenitor cells, thrombin and phorbol esters potentiate cellular cAMP formation in response to Gs-coupled receptor agonists such as prostaglandin E1 (PGE1). We show here that the two agonists achieve their phenotypically similar effects by using distinctly different signaling pathways, both of which require protein kinase C (PKC) activation. After short term exposure (11 min), phorbol esters caused an alkaline shift of cellular pH by approximately 0.1 unit, resulting in a 1.5-2-fold increase in PGE1-induced cAMP formation. The effect of phorbol esters was inhibited by 5-(N-ethyl-N-isopropyl)amiloride, a specific inhibitor of the Na+/H+ exchanger, and by the PKC inhibitors GF 109203X, G? 6976, and staurosporine. Thrombin increased cellular pH by only 0.02-0.05 unit but seemed to potentiate PGE1-stimulated cAMP formation by an effect on the Gs-activated adenylyl cyclase involving a Ca2+-independent (novel) PKC. This effect was inhibited by GF 109203X and staurosporine but was resistant to 5-(N-ethyl-N-isopropyl)amiloride or G? 6976. Inactivation of PKC by incubation of the cells in the presence of 10 nM phorbol-12-myristate-13-acetate for 18 hr completely abolished the potentiating effect of thrombin on cyclase activity, whereas the pH-dependent stimulation was fully retained. Northern blots with specific cDNA probes and a lack of Ca2+ sensitivity indicate that progenitor cells predominantly express adenylyl cyclase type VII. Our results suggest that in normal human erythroid progenitors, thrombin can activate pH-dependent and -independent, PKC-linked pathways converging on adenylyl cyclase type VII to potentiate cAMP formation in response to Gs-coupled receptor agonists.