Neurohumoral Agonists Research Articles

Activation of NFATc1 is directly mediated by IP3 in adult cardiac myocytes

The Ca(2+)-sensitive nuclear factor of activated T cell (NFAT) transcription factors are implicated in cardiac development and cellular remodeling associated with cardiac disease. In adult myocytes it is not resolved what specific Ca(2+) signals control the activity of different NFAT isoforms in an environment that undergoes large changes of intracellular Ca(2+) concentration with every heart beat. Cardiac myocytes possess the complete inositol 1,4,5-trisphosphate (IP(3))/Ca(2+)-signaling cassette; however, its physiological and pathological significance has been a matter of ongoing debate. Therefore, we tested the hypothesis whether IP(3) receptor activation regulates NFAT activity in cardiac myocytes. We used confocal microscopy to quantify the nuclear localization of NFATc1-green fluorescent protein (GFP) and NFATc3-GFP fusion proteins (quantified as the ratio of nuclear NFAT to cytoplasmic NFAT) in response to stimulation with neurohumoral agonists. In rabbit atrial myocytes, an overnight stimulation with endothelin-1, angiotensin II, and phenylephrine induced nuclear accumulation of NFATc1 that was sensitive to calcineurin inhibitors (cyclosporin A or inhibitor of NFAT-calcineurin association-6) and prevented by the IP(3) receptor inhibitor 2-aminoethoxydiphenyl borate. Furthermore, a direct elevation of intracellular IP(3) with a cell-permeable IP(3) acetoxymethyl ester (10 μM) induced nuclear localization of NFATc1. With a fluorescence-based in vivo assay, we showed that endothelin-1 also enhanced the transcriptional activity of NFATc1 in atrial cells. The agonists failed to activate NFATc1 in rabbit ventricular cells, which express IP(3) receptors at a lower density than atrial cells. They also did not activate NFATc3, an isoform that is highly influenced by nuclear export processes, in both cell types. Our data show that the second messenger IP(3) is directly involved in the activation of NFATc1 in adult atrial cardiomyocytes.

Biomechanical and neurohumoral stimulation of neonatal rat ventricular myocytes induce focal adhesion kinase phosphorylation at S910

Biomechanical and neurohumoral stimulation of cardiomyocytes rapidly increase focal adhesion kinase (FAK) phosphorylation at tyrosine residues, but little is known about FAK phosphorylation at serine residues. Stimulation of cultured neonatal rat ventricular myocytes (NRVM) by 20% static stretch promoted a 4‐5 fold increase in FAK S910 phosphorylation, but not S722 phosphorylation, as revealed by Western blotting with phosphospecific antibodies. The neurohumoral agonists endothelin‐1, angiotensin II, and phenylephrine also stimulated FAK S910 phosphorylation. Direct activation of protein kinase C isoforms with phorbol 12‐myristate 13‐acetate (PMA) significantly increased FAK S910 phosphorylation, whereas the PKC inhibitor GF102903X, but not Gö6983 prevented ET‐1‐induced FAK S910 phosphorylation. These results suggest that both stimulation increase FAK S910 phosphorylation via novel, but not classical PKCs. Treatment with the MEK1 inhibitors U0126 or PD98059 also inhibited FAK S910 phosphorylation in response to stretch and ET‐1, whereas the Src inhibitor PP2 reduced FAK S910 phosphorylation induced by ET‐1, but not by stretch. Furthermore, inhibition of ILK by shRNA‐mediated knockdown prevented stretch‐induced FAK S910 phosphorylation. Thus, these data demonstrate that biomechanical and neurohumoral stimuli stimulate NRVM FAK S910 phosphorylation via different signaling pathways.

Loss of myocardial LIF receptor in experimental heart failure reduces cardiotrophin-1 cytoprotection. A role for neurohumoral agonists?

Cardiomyocyte loss is involved in the transition from compensatory left ventricular hypertrophy (LVH) to heart failure (HF). Our aim was to investigate the status of the leukaemia inhibitory factor receptor (LIFR)/gp130 survival pathway and its cytoprotective activity in intact cardiac tissue and in cardiomyocytes obtained from adult spontaneously hypertensive rats (SHR) with LVH (non-failing SHR) and from aged SHR with overt HF (failing SHR). Cardiac morphometry was assayed by planimetry in an image analysis system. mRNA and protein expression were quantified by real time RT-PCR and Western blotting. Receptors were localized by immunocytochemistry. Trypan blue staining, TUNEL, and MTT cell viability assays were employed to study the cytoprotective activity of cardiotrophin-1 (CT-1) in isolated caridomyocytes. Compared to non-failing SHR, failing SHR exhibited enhanced myocardial cell death (p<0.01) demonstrated by the increase in Bax/Bcl-2 ratio, caspase-3 activation and poly (ADP-ribose) polymerase (PARP) fragmentation. Failing SHR had a 7-fold diminished expression (p<0.01) of LIFR, no changes in gp130, and 1.6-fold increased myocardial expression (p<0.01) of CT-1. In cardiomyocytes isolated from non-failing SHR, recombinant CT-1 inhibited apoptotic and non-apoptotic cell death induced by angiotensin II or hydrogen peroxide. LIFR protein was entirely absent in cardiomyocytes isolated from failing SHR, which were resistant to the cytoprotective effects of CT-1. Finally, stimulation of non-failing SHR cardiomyocytes with angiotensin II, aldosterone, norepinephrine or endothelin-1 significantly decreased (p<0.01) LIFR expression. These data suggest that loss of CT-1-dependent survival mechanisms may contribute to the increase of cell death associated with HF in SHR. Neurohumoral activation may contribute to this alteration via suppression of LIFR.

Oxidative Stress and Growth-Regulating Intracellular Signaling Pathways in Cardiac Myocytes

The toxic effects of oxidative stress on cells (including cardiac myocytes, the contractile cells of the heart) are well known. However, an increasing body of evidence has suggested that increased production of reactive oxygen species (ROS) promotes cardiac myocyte growth. Thus, ROS may be 'second messenger' molecules in their own right, and growth-promoting neurohumoral agonists might exert their effects by stimulating production of ROS. The authors review the principal growth-promoting intracellular signaling pathways that are activated by ROS in cardiac myocytes, namely the mitogen-activated protein kinase cascades (extracellular signal-regulated kinases 1/2, c-Jun N-terminal kinases, and p38-mitogen-activated protein kinases) and the phosphoinositide 3-kinase/protein kinase B (Akt) pathway. Possible mechanisms are discussed by which these pathways are activated by ROS, including the oxidation of active site cysteinyl residues of protein and lipid phosphatases with their consequent inactivation, the potential involvement of protein kinase C or the apoptosis signal-regulating kinase 1, and the current models for the activation of the guanine nucleotide binding protein Ras.

Nitric Oxide Not Carbon Monoxide Mediates Nonadrenergic Noncholinergic Relaxation in the Murine Internal Anal Sphincter

Inhibitory reflexes in the internal anal sphincter (IAS) are controlled by inhibitory nonadrenergic, noncholinergic innervation (i-NANC). We investigated the roles of 3 different neurohumoral agonists as possible i-NANC neurotransmitters: carbon monoxide (CO), nitric oxide (NO), and vasoactive intestinal peptide (VIP). IAS smooth muscle strips were isolated from wild-type (WT), heme oxygenase (HO)-2 knockout (HO-2-/-) and neuronal NO synthase (nNOS) knockout (nNOS-/-) mice. Relaxation of IAS was induced by CO, NO, VIP, and electrical field stimulation (EFS) in the presence and absence of neurohumoral inhibitors (tin protoporphyrin IX [SnPP IX] for CO synthesis, N(omega)-nitro-L-arginine [L-NNA] for NO synthesis, and VIP(10-28) for VIP receptor). Western blot and immunohistochemistry were used to test the presence and localization of HO (for CO synthesis) types 1 (HO-1) and 2 (HO-2), neuronal NO synthase (nNOS, for NO synthesis), and VIP. All 3 neurohumoral agonists produced relaxation (with no difference between WT and HO-2-/- IAS), but CO was over 100 times less potent than NO and VIP. EFS produced relaxation in WT and HO-2-/- IAS with the same intensity. L-NNA and nNOS deletion (approximately 80%) and VIP(10-28) (approximately 15%) significantly inhibited the relaxations, whereas SnPP IX had no effect. Positive immunoreactivities for HO-2, nNOS, and VIP were found in the myenteric plexus of WT IAS. HO-2-/- IAS did not express immunoreactivity for HO-2. i-NANC relaxations of mouse IAS are primarily mediated via NO (by nNOS activity) and partly via VIP. CO directly relaxes the mouse IAS but does not play any significant role in the i-NANC relaxation.

Determinants of inducible brain natriuretic peptide promoter activity

Atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) are polypeptide hormones belonging to the cardiac-derived mammalian natriuretic peptide system. These hormones share the same biological properties and receptors and both play important roles in the maintenance of fluid and electrolyte balance and in cardiovascular growth. Most hemodynamic and neurohumoral stimuli can coordinately increase ANF and BNP gene expression. However, instances of discoordinated ANF and BNP gene expression have been described, providing an opportunity for investigating the mechanisms that differentially regulate the expression of the natriuretic peptide genes. For example, exposure of cardiocytes in culture to certain pro-inflammatory cytokines and conditioned medium from mixed lymphocyte cultures upregulate BNP but not ANF gene expression. BNP promoter activity is also upregulated under these conditions but the cis-acting elements involved in this phenomenon are not known. In comparison to the ANF gene, less is known about BNP promoter consensus elements that regulate gene expression by mechanical or neurohumoral agonists. A number of cis-acting elements for GATA, Nkx2.5, NF-κB and TEF transcription factors have recently been identified within the BNP promoter that regulate BNP expression in response to specific agonists. This review focuses on the information available regarding cis-acting determinants responsible for inducible BNP transcription.

Morphology of Isolated Colonic Crypts

The traditional paradigm of colonic fluid and electrolyte transport includes a spatial separation of absorptive and secretory processes to surface and crypt cells, respectively. Recent studies of isolated microperfused colonic crypts revealed constitutive Na-dependent fluid absorption while secretion is regulated by one or more neurohumoral agonists. One obvious reason for the difference found in microdissected crypts is their separation from the lamina propria milieu. While it has been shown that isolated crypts are devoid of obvious lamina propria elements, including pericryptal fibroblasts, detailed morphologic information of the content of isolated crypts has been lacking. To characterize the morphology of the isolated crypt, we performed transmission electron microscopy (TEM) and immunofluorescence on microdissected and Ca²⁺ chelated crypts. Crypt cell type analysis was carried out separately on intact rat colon using light microscopy. TEM revealed a complete lack of either lamina propria cells or extracellular material in crypts isolated by either technique. TEM also revealed a subtle difference between the two isolation methods, with intact basal membranes in microdissected crypts but focal disruption of basal membranes in Ca²⁺- chelated crypts. Immunofluorescent stains for two basement membrane components (laminin and collagen type IV) revealed the presence of adherent basement membrane only on microdissected crypts; evidence that the plane of separation differs in these two preparations. Crypt cell type analysis on intact rat colon revealed an equal proportion of goblet cells in the right and left colon (approximately 50%) when measuring the middle 70% of the crypts – the area studied during crypt microperfusion. This morphologic analysis will increase our understanding of the observed physiology of isolated colonic crypts.

Cl-dependent Na-H exchange. A novel colonic crypt transport mechanism.

This communication summaries a series of observations of the transport function of the crypt of the rat distal colon. Development of methods to study both 22Na uptake by apical membrane vesicles prepared from crypt cells and intracellular pHi (pHi), fluid movement (Jv), and bicarbonate secretion during microperfusion of the crypt has led to the identification of (1) a novel Cl-dependent Na-H exchange (Cl-NHE) that most likely represents the coupling of a Cl channel to a Na-H exchange isoform that has not as yet been identified and (2) bicarbonate secretion that appears to be most consistent with HCO3 uptake across the basolateral membrane by a mechanism that is closely linked to Cl transport and its movement across the apical membrane via an anion channel. Na-dependent fluid absorption is the constitutive transport process in the crypt, while fluid secretion is regulated by one or more neurohumoral agonists. Cl-NHE is responsible for both the recovery/regulation of pHi in crypt cells to an acid load and fluid absorption.

Glucose-induced insulin secretion from islets of fasted rats: modulation by alternate fuel and neurohumoral agonists.

Islets from fed and 24-h-fasted rats were studied immediately after collagenase isolation. (1) After a 24-h fast, the insulin secretory responses to 8 mM glucose measured during perifusion were reduced by more than 90% from islets of fasted donors. (2) Increasing glucose to 11 or 27.5 mM resulted in enhanced insulin secretion from islets of fasted animals. (3) Fasting did not reduce islet insulin content. (4) Responses to 8 or 27.5 mM glucose were not affected if fatty acid-free albumin was used during the perifusion. (5) Inclusion of alpha-ketoisocaproate (5 mM), monomethyl succinate (10 mM) or carbachol (10 microM) significantly amplified insulin release from fasted islets in the simultaneous presence of 8 mM glucose. (6) Phospholipase C activation by glucose, carbachol or their combination was not adversely affected by fasting. (7) The response to the protein kinase C activator, phorbol 12-myristate 13-acetate (500 nM), was reduced by about 60% after fasting. (8) Extending the fast to 48 h resulted in a severe decline in response to 11 mM glucose; however, the further addition of 10 microM carbachol still enhanced release from these islets. The results confirm that caloric restriction impairs islet sensitivity to glucose stimulation and that protein kinase C may be involved in the reduction of glucose-induced insulin release from these islets. The activation of phospholipase C by cholinergic stimulation may contribute to the maintenance of insulin secretion from calorically restricted animals. These results also demonstrate that free fatty acids are not essential for glucose to evoke secretion from isolated islets of fasted donors.

Novel Transport Properties of Colonic Crypt Cells: Fluid Absorption and Cl-Dependent Na-H Exchange

Colonic ion transport is heterogeneous including the long-accepted spatial separation of absorptive and secretory processes between surface and crypt cells. We recently described the isolation of individual crypts from the rat distal colon that were studied using microperfusion technology. Na-dependent fluid absorption was consistently demonstrated in these crypts during perfusion with a Ringer-like solution; dibutyryl cyclic AMP, VIP and acetylcholine, when added to the bath solution, all induced net fluid secretion. As several morphologic techniques, including immunocytochemistry, failed to provide evidence for the presence of myofibroblasts in the isolated crypt preparation, we propose that a Na-dependent absorptive process is a constitutive transport mechanism in crypt cells, while secretory processes are regulated by the release of one or more neurohumoral agonists from lamina propria cells including myofibroblasts. The mechanism of Na-dependent fluid movement was also studied by determining [H] gradient stimulation of 22Na uptake in isolated apical membrane vesicles (AMV) from crypt cells. In contrast to Na-H exchange in surface cell AMV, Na-H exchange in crypt cells is Cl-dependent. Intracellular pH determined in crypt cells using video-imaging fluorescence microscopy established that the response to an acid load requires both lumen Na and Cl. As a result, these studies have identified a novel Cl-dependent Na-H exchange in crypt AMV that may mediate apical membrane Na uptake and regulate pHi.

Signal transduction in pancreatic beta-cells: regulation of insulin secretion by information flow in the phospholipase C/protein kinase C pathway.

The physiologic regulation of glucose-induced insulin secretion is dependent upon the activation of information flow in the phospholipase C (PLC)/protein kinase C (PKC) signal transduction system. In both rat and human pancreatic beta-cells, glucose has several time-dependent effects on secretory responsiveness including the regulation of biphasic insulin secretion, time-dependent potentiation and time-dependent suppression. PLC/PKC activation has been implicated in all three response patterns. Islets of Langerhans contain the three major PLC isozyme classes (beta1, gamma1 and delta1) and the available evidence suggests that one class is activated by fuel secretagogues and another by neurohumoral agonists. The expression and activation of PLC is labile. When rat islet are cultured for short periods, the content and activation of PLC in response to glucose decreases and this biochemical defect in signal transduction is paralleled by significant reductions in glucose-induced insulin secretion. Similar defects are observed when human islets are cultured as well. Mouse islet responses to glucose stimulation differ in several major and dramatic ways from rat and human islet responses. When stimulated by 15mM glucose, mouse islets fail to develop a rising second phase secretory response, they fail to exhibit either time-dependent potentiation or time-dependent suppression to the hexose. Biphasic insulin secretion can be evoked and time-dependent potentiation can be induced in mouse islets by carbachol, an agonist that activates an isozyme of PLC distinct from that activated by glucose. These divergent response patterns are attributable to the underexpression in mouse islets, when compared to rat islets, of a fuel-sensitive PLC isozyme. When taken in their entirety, the experimental evidence suggests that the activation of PLC is an essential component in the physiologic regulation of insulin secretion and that disordered activation of the enzyme culminates in disordered insulin release.

Effect of neuromimetics upon the release of atrial natriuretic peptide granules: Are multiple pathways involved in secretion?

The release of atrial natriuretic peptide (ANP) in response to the application of neurohumoral agonists (neuromimetics) is directly demonstrated and quantified at the cellular level, using an ultrastructural assay developed to quantify secretion. The assay uses an in situ tannic acid perfusion technique to arrest the exocytosis of atrial secretory granules in the anesthetized rat. The animal is perfused with the neuromimetic, and secretory granules, which retain the capacity to undergo exocytosis throughout the subsequent 30 min tannic acid perfusion, accumulate at the cell surface in a state of fusion with the plasma membrane. Quantification of arrested granules thus provides a measure of the rate of granule release and allows the responses to different agents to be assessed. The actions of three different agents were investigated: isoproterenol, phenylephrine, and acetylcholine. In previously published studies, investigations of the actions of these agents on ANP release has produced unclear and sometimes contradictory results. Using our ultrastructural assay, it was found that during the 30 min perfusion period neither isoprenaline nor phenylephrine caused a significant change in the rate of secretory granule release, whereas acetylcholine significantly decreased the rate of granule release. A new model of secretion is proposed to integrate these findings with previous results and help clarify the complex picture of atrial natriuretic peptide release.

Fluid absorption in isolated perfused colonic crypts.

A spatial segregation of ion transport processes between crypt and surface epithelial cells is well-accepted and integrated into physiological and pathophysiological paradigms of small and large intestinal function: Absorptive processes are believed to be located in surface (and villous) cells, whereas secretory processes are believed to be present in crypt cells. Validation of this model requires direct determination of fluid movement in intestinal crypts. This study describes the adaptation of techniques from renal tubule microperfusion to hand-dissect and perfuse single, isolated crypts from rat distal colon to measure directly fluid movement. Morphologic analyses of the isolated crypt preparation revealed no extraepithelial cellular elements derived from the lamina propria, including myofibroblasts. In the basal state, crypts exhibited net fluid absorption (mean net fluid movement = 0.34 +/- 0.01 nl.mm-1.min-1), which was Na+ and partially HCO3- dependent. Addition of 1 mM dibutyryl-cyclic AMP, 60 nM vasoactive intestinal peptide, or 0.1 mM acetylcholine to the bath (serosal) solution reversibly induced net fluid secretion (net fluid movement approximately -0.35 +/- 0.01 nl.mm-1.min-1). These observations permit speculation that absorption is a constitutive transport function in crypt cells and that secretion by crypt cells is regulated by one or more neurohumoral agonists that are released in situ from lamina propria cells. The functional, intact polarized crypt described here that both absorbs and secretes will permit future studies that dissect the mechanisms that govern fluid and electrolyte movement in the colonic crypt.

Synergistic interaction of glucose and neurohumoral agonists to stimulate islet phosphoinositide hydrolysis

The interaction between neurohumoral agonists and glucose to stimulate phosphoinositide (PI)-specific phospholipase C (PLC) and insulin release was examined. In freshly isolated rat islets, maximal glucose (40 mM), cholecystokinin (CCK; 300 nM), or carbachol (CCh; 1 mM) stimulated PI hydrolysis 6.5-, 9.8-, and 5.7-fold, respectively, above basal. The combination of glucose and CCK or of glucose and CCh, but not of CCK and CCh, synergistically increased PI hydrolysis 23.2- and 21.6-fold, respectively, indicating that these secretagogues activate PLC by distinct pathways and that there is an interaction between them. This synergy was maximal at physiological concentrations of stimulatory glucose (8-10 mM) and was paralleled by a marked synergistic stimulation of insulin secretion. The enhanced PI response was partially Ca2+ dependent and may involve the activation of distinct isozymes of PLC, which we identify in islets. These studies demonstrate for the first time a unique and highly sensitive synergistic interaction between glucose and neurohumoral agonists to stimulate PI hydrolysis, and they suggest that enhanced PI hydrolysis is important in the potentiation of glucose- and neurohumoral-stimulated insulin secretion.

Metabolic activation of Ca(2+)-independent phosphoinositide hydrolysis in beta-cells and its role in the control of insulin secretion.

Recent studies have led to the proposal that the oxidative metabolism of glucose leads to the generation of messengers, in addition to ATP, that are important in the ability of changes in extracellular glucose concentration to stimulate insulin secretion from pancreatic beta-cells. In particular, there is now evidence that glucose induces both a Ca(2+)-dependent and Ca(2+)-independent increase in phosphoinositide (PI) hydrolysis. To explore the relationship between oxidative metabolism and PI hydrolysis, we examined the effect of low concentrations (2.5 mM) of alpha-ketoisocaproate (KIC) and monomethylsuccinate (MMSucc) either alone or in combination on insulin secretion and PI hydrolysis in isolated rat pancreatic islets incubated with either no glucose, 5 mM glucose, or 20 mM glucose. A combination of KIC and MMSucc leads to a marked increase in largely (80%) Ca(2+)-independent PI hydrolysis in either the absence or presence of 5 mM glucose. When glucose is absent, this combination of substrates induces a very small and transient first phase of insulin secretion but no significant second phase of secretion. In the presence of 5 mM glucose, either KIC or MMSucc alone induces a first phase of insulin secretion with a peak secretory rate 10-fold greater than the basal rate but only a small second phase of secretion approximately 5-fold above control. However, in the presence of 5 mM glucose, the combination of KIC plus MMSucc induces a large biphasic increase in insulin secretion: peak first-phase secretion is increased 30-fold, and second-phase 40-fold. These response are comparable to those induced by 20 mM glucose and are completely inhibited by 0.5 microM nitrendipine. In contrast, KIC plus MMSucc do not enhance the insulin secretory response induced by 20 mM glucose. Previous data showed that when 20 mM glucose acts, the resulting increase in PI hydrolysis is only partially Ca2+ dependent. A reanalysis of these data shows that raising the glucose concentration from 5 to 7 mM causes a 2-fold increase in Ca(2+)-independent PI hydrolysis, and a further increase to 20 mM leads to a further 2-fold increase in Ca(2+)-dependent PI hydrolysis. These data show that these two pathways are regulated by different ranges of glucose concentration. They raise the interesting possibility that these distinct pathways have different signaling functions. In particular, raising the glucose concentration from 5 to 7 mM is known to alter the responsiveness of beta-cells to a variety of neurohumoral agonists and to tolbutamide.(ABSTRACT TRUNCATED AT 400 WORDS)

Metabolic activation of Ca(2+)-independent phosphoinositide hydrolysis in beta-cells and its role in the control of insulin secretion

Recent studies have led to the proposal that the oxidative metabolism of glucose leads to the generation of messengers, in addition to ATP, that are important in the ability of changes in extracellular glucose concentration to stimulate insulin secretion from pancreatic beta-cells. In particular, there is now evidence that glucose induces both a Ca(2+)-dependent and Ca(2+)-independent increase in phosphoinositide (PI) hydrolysis. To explore the relationship between oxidative metabolism and PI hydrolysis, we examined the effect of low concentrations (2.5 mM) of alpha-ketoisocaproate (KIC) and monomethylsuccinate (MMSucc) either alone or in combination on insulin secretion and PI hydrolysis in isolated rat pancreatic islets incubated with either no glucose, 5 mM glucose, or 20 mM glucose. A combination of KIC and MMSucc leads to a marked increase in largely (80%) Ca(2+)-independent PI hydrolysis in either the absence or presence of 5 mM glucose. When glucose is absent, this combination of substrates induces a very small and transient first phase of insulin secretion but no significant second phase of secretion. In the presence of 5 mM glucose, either KIC or MMSucc alone induces a first phase of insulin secretion with a peak secretory rate 10-fold greater than the basal rate but only a small second phase of secretion approximately 5-fold above control. However, in the presence of 5 mM glucose, the combination of KIC plus MMSucc induces a large biphasic increase in insulin secretion: peak first-phase secretion is increased 30-fold, and second-phase 40-fold. These response are comparable to those induced by 20 mM glucose and are completely inhibited by 0.5 microM nitrendipine. In contrast, KIC plus MMSucc do not enhance the insulin secretory response induced by 20 mM glucose. Previous data showed that when 20 mM glucose acts, the resulting increase in PI hydrolysis is only partially Ca2+ dependent. A reanalysis of these data shows that raising the glucose concentration from 5 to 7 mM causes a 2-fold increase in Ca(2+)-independent PI hydrolysis, and a further increase to 20 mM leads to a further 2-fold increase in Ca(2+)-dependent PI hydrolysis. These data show that these two pathways are regulated by different ranges of glucose concentration. They raise the interesting possibility that these distinct pathways have different signaling functions. In particular, raising the glucose concentration from 5 to 7 mM is known to alter the responsiveness of beta-cells to a variety of neurohumoral agonists and to tolbutamide.

Physiology and Pathophysiology of Insulin Secretion

Mechanisms by which various classes of extracellular signals regulate insulin secretion are discussed regarding their cellular and molecular actions. Under physiological circumstances, the small postprandial changes in plasma glucose concentrations (approximately 4.4-6.6 mM) primarily serve as a conditioned modifier of insulin secretion and dramatically alter the responsiveness of islets to a combination of neurohormonal agonists. These agonists have two functions. Cholecystokinin (CCK) and acetylcholine activate the hydrolysis of polyphosphoinositides, and gastric inhibitory polypeptide (GIP) and glucagonlike peptide 1 activate adenylate cyclase. These two functional classes of neurohumoral agonists act synergistically to enhance insulin secretion when plasma glucose is greater than 6.0 mM but not when it is less than or equal to 4 mM. On the other hand, an increase in plasma glucose concentration to 8-10 mM induces an increase in insulin secretory rate in the absence of any of the neurohormonal agonists. Remarkably, high glucose leads to an increase in the same intracellular signals, as does a combination of acetylcholine and GIP. On the basis of these data, a model of how insulin secretion is regulated under physiological circumstances is proposed. This model emphasizes that the regulation of insulin secretion occurs in three stages: cephalic, early enteric, and later enteric. In this view, the crucial event occurring during the first two phases is the agonist-induced, translocation of protein kinase C (PKC) to the plasma membrane under conditions in which an increase in Ca2+ influx does not occur. PKC is now in a cellular location and a Ca2(+)-sensitive conformation such that an increase in Ca2+ influx rate occurring during the third phase leads to its immediate activation and an enhanced rate of insulin secretion. Furthermore, under physiological circumstances, an optimal insulin secretory response is dependent on a correct temporal pattern of signals arising from neural and enteric sources. If this pattern is deranged, an abnormal pattern of insulin secretion is observed. An important new insight is provided by the observation that agonists (e.g., CCK or acetylcholine) that act to stimulate the hydrolysis of phosphatidylinositides, when acting for a short period (10-20 min), induce an enhanced responsiveness of islets to glucose, i.e., proemial sensitization. However, when acting unopposed for several hours, these agonists will induce a time-dependent suppression of responsiveness to glucose and other agonists. The latter observation implies that optimal insulin secretion is dependent on periodic rather than a continuous exposure to the correct pattern of extracellular signals.(ABSTRACT TRUNCATED AT 400 WORDS)

Mechanisms of hormonal regulation of hepatic glucose metabolism.

Acute hormonal regulation of liver carbohydrate metabolism mainly involves changes in the cytosolic levels of cAMP and Ca2+. Epinephrine, acting through beta 2-adrenergic receptors, and glucagon activate adenylate cyclase in the liver plasma membrane through a mechanism involving a guanine nucleotide-binding protein that is stimulatory to the enzyme. The resulting accumulation of cAMP leads to activation of cAMP-dependent protein kinase, which, in turn, phosphorylates many intracellular enzymes involved in the regulation of glycogen metabolism, gluconeogenesis, and glycolysis. These are (1) phosphorylase b kinase, which is activated and, in turn, phosphorylates and activates phosphorylase, the rate-limiting enzyme for glycogen breakdown; (2) glycogen synthase, which is inactivated and is rate-controlling for glycogen synthesis; (3) pyruvate kinase, which is inactivated and is an important regulatory enzyme for glycolysis; and (4) the 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase bifunctional enzyme, phosphorylation of which leads to decreased formation of fructose 2,6-P2, which is an activator of 6-phosphofructo-1-kinase and an inhibitor of fructose 1,6-bisphosphatase, both of which are important regulatory enzymes for glycolysis and gluconeogenesis. In addition to rapid effects of glucagon and beta-adrenergic agonists to increase hepatic glucose output by stimulating glycogenolysis and gluconeogenesis and inhibiting glycogen synthesis and glycolysis, these agents produce longer-term stimulatory effects on gluconeogenesis through altered synthesis of certain enzymes of gluconeogenesis/glycolysis and amino acid metabolism. For example, P-enolpyruvate carboxykinase is induced through an effect at the level of transcription mediated by cAMP-dependent protein kinase. Tyrosine amino-transferase, serine dehydratase, tryptophan oxygenase, and glucokinase are also regulated by cAMP, in part at the level of specific messenger RNA synthesis. The sympathetic nervous system and its neurohumoral agonists epinephrine and norepinephrine also rapidly alter hepatic glycogen metabolism and gluconeogenesis acting through alpha 1-adrenergic receptors. The primary response to these agonists is the phosphodiesterase-mediated breakdown of the plasma membrane polyphosphoinositide phosphatidylinositol 4,5-P2 to inositol 1,4,5-P3 and 1,2-diacylglycerol. This involves a guanine nucleotide-binding protein that is different from those involved in the regulation of adenylate cyclase. Inositol 1,4,5-P3 acts as an intracellular messenger for Ca2+ mobilization by releasing Ca2+ from the endoplasmic reticulum.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenylate cyclase responses to neurohumoral agonists in microvascular elements of the rabbit brain.

Adenylate cyclase responses to neurohumoral agonists in microvascular elements of the rabbit brain.

Cyclic nucleotide systems in the microcirculation of mammalian brain.

The cellular elements of the central microcirculation, namely the capillaries, choroid plexus and pia, contain adrenergic nerve endings whose functions may be involved in the regulation of permeability (1–6). An important aspect of noradrenergic action is stimulation of the receptor enzyme, adenylate cyclase with concomitant formation of adenosine cyclic 3′,5′-monophosphate (cyclic AMP). Recent work that I did with co-investigators (7–10) and the works of others (11–15) showed that the cellular components of the microcirculation contain active adenylate cyclase systems capable of responding to a variety of neurohumoral agonists, including norepinephrine. Joō (16) found that an infusion of dibutyryl cyclic AMP yielded an enhanced number of pinocytotic vesicles in mouse cerebral capillaries, and suggested that the cyclic nucleotide has a role in central nervous system transport mechanisms. In this chapter I will discuss our previous work and present new data characterizing the specific nature of the receptor enzymes in rat and rabbit cerebral microvessels. In addition, since the cerebral capillaries develop altered permeability in certain pathologic conditions such as hypertension (6,17–19), we will report changes seen in the cyclase system of cerebral capillaries from the spontaneously hypertensive rats (SHR).KeywordsAdenylate CyclaseChoroid PlexusCyclic NucleotideBrain CapillaryPinocytotic VesicleThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.