Type Voltage-dependent Calcium Channel Research Articles

Cdk5 Sebagai Target Potensial Penemuan Obat Anti Nyeri Kronik

Chronic pain is a very disturbing condition and to date, no effective and safe medicine has been found. This could be caused by existing drugs not targeting the key factor in pain transmission. Cdk5 is a protein kinase that has been shown to have a key role in the transmission of chronic pain. These important roles include: activation of TRPV1, NMDA, P/Q type voltage-dependent calcium channels, and ATP-gate P2X receptors. Cdk5 could be used as a potential target in the development of new anti-chronic pain drugs. Several aspects of concern in developing this drugs are the role of Cdk5 in physiological and pathological conditions because it’s will be related to the risk of side effects. Another factor that really needs to be considered is the location of Cdk5 expression because it will related to the drug design. Several drugs that have been developed as Cdk5 inhibitors are roscovitin, olomoucine, and indirubin.

Voltage Dependent N Type Calcium Channel in Mouse Egg Fertilization.

Repetitive changes in the intracellular calcium concentration ([Ca2+]i) triggers egg activation, including cortical granule exocytosis, resumption of second meiosis, block to polyspermy, and initiating embryonic development. [Ca2+]i oscillations that continue for several hours, are required for the early events of egg activation and possibly connected to further development to the blastocyst stage. The sources of Ca2+ ion elevation during [Ca2+]i oscillations are Ca2+ release from endoplasmic reticulum through inositol 1,4,5 tri-phosphate receptor and Ca2+ ion influx through Ca2+ channel on the plasma membrane. Ca2+ channels have been characterized into voltage-dependent Ca2+ channels (VDCCs), ligand-gated Ca2+ channel, and leak-channel. VDCCs expressed on muscle cell or neuron is specified into L, T, N, P, Q, and R type VDCs by their activation threshold or their sensitivity to peptide toxins isolated from cone snails and spiders. The present study was aimed to investigate the localization pattern of N and P/Q type voltage-dependent calcium channels in mouse eggs and the role in fertilization. [Ca2+]i oscillation was observed in a Ca2+ contained medium with sperm factor or adenophostin A injection but disappeared in Ca2+ free medium. Ca2+ influx was decreased by Lat A. N-VDCC specific inhibitor, ω-Conotoxin CVIIA induced abnormal [Ca2+]i oscillation profiles in SrCl2 treatment. N or P/Q type VDC were distributed on the plasma membrane in cortical cluster form, not in the cytoplasm. Ca2+ influx is essential for [Ca2+]i oscillation during mammalian fertilization. This Ca2+ influx might be controlled through the N or P/Q type VDCCs. Abnormal VDCCs expression of eggs could be tested in fertilization failure or low fertilization eggs in subfertility women.

Saffron Induced Relaxation in Isolated Rat Aorta via Endothelium Dependent and Independent Mechanisms.

Crocus sativus L. (saffron) is a widely used food additive for its color and taste. The hypotensive effects of saffron have been shown in previous studies. The aim of this study was to evaluate the mechanism of vasodilatory effects induced by saffron on isolated rat aorta. To study the vasodilatory effects of saffron aqueous extract (0.5, 1 and 2 mg/mL), isolated rat thoracic aorta rings were contracted by 10-6 M phenylephrine (PE) or KCl 80 mM. The vasodilatory effect of saffron was also evaluated both on intact and denuded endothelium aortic rings. To study the role of nitric oxide and prostacyclin in relaxation induced by saffron, aortic rings were incubated by L-NAME (10-6 M) and indomethacin (10-5 M) respectively for 20 min. Saffron induced relaxation in endothelium-intact aortic rings precontracted with PE in a concentration dependent manner. The obtained relaxation induced by the highest saffron concentration in endothelium-intact aortic rings precontracted with KCl was less than that observed in endothelium-intact aortic rings precontracted with PE. The relaxant activity of saffron was abolished by incubation of aortic rings with L-NAME but not in the presence of indomethacin. Also, the vasodilatory activity of saffron was partially abolished in endothelium denuded aortic rings. Saffron induced relaxation in isolated rat aortic rings might be due particularly to its effect on endothelium via nitric oxide synthase pathway and partly due to the effect on vascular smooth muscle cells via L type voltage dependent calcium channels.

PP.31.05] ALTERED CALCIUM SENSITIZATION IN HYPERTENSIVE REN-2 TRANSGENIC RATS

Objective: A comparison of BP response to Ca channel opener BAY K8644 prior and after Rho kinase inhibition indicated decreased calcium sensitization in spontaneously hypertensive rats (SHR), although blood pressure (BP) response of intact SHR to Rho kinase inhibitor fasudil was enhanced (Behuliak et al. J Hypertens 31: 2025, 2013). The aim of the present study was to evaluate calcium sensitization in hypertensive transgenic rats with murine Ren-2 gene (TGR) and their normotensive HanSD controls using the same methods. We also tried to elucidate the interference of baroreflex-activated sympathetic activity with BP response to fasudil. Design and method: Blood pressure and heart rate responses to increasing doses BAY K8644 (opener of L type voltage-dependent calcium channels) was determined in conscious rats (subjected to acute RAS and SNS blockade). BP response was recorded prior and after Rho kinase inhibition by fasudil and calcium sensitization was estimated from the difference between both BP response curves. Furthermore, BP-lowering effects of acute administration of increasing fasudil doses were measured in conscious intact rats as well as in rats subjected to combined blockade of RAS, SNS and NOS in which baroreflex efficiency was suppressed. Results: Using the above BAY K8644-based approach we demonstrated moderate attenuation of calcium sensitization in hypertensive TGR. We also observed enhanced acute BP response to fasudil in intact TGR compared to HanSD rats. This different BP response was accompanied by more pronounced heart rate increase in HanSD than in TGR, indicating attenuated baroreflex efficiency in hypertensive TGR. If baroreflex operation was minimized by ganglionic blockade in rats subjected to combined RAS, SNS and NO synthase blockade, fasudil-induced BP fall was manifold augmented in HanSD but unchanged in TGR, suggesting the importance of baroreflex-activated sympathetic activity in compensation of fasudil-induced BP changes. Conclusions: Calcium sensitization is attenuated in hypertensive TGR compared to normotensive controls, but this difference can be seen only in the absence of operating sympathetic nervous system which compensates BP changes elicited by acute Rho kinase inhibition much better in normotensive than in hypertensive animals. Partially supported by grant AZV 15–25396A (MH CR)

OS 05-03 DECREASED RHO-KINASE-BASED CALCIUM SENSITIZATION IN HYPERTENSIVE REN-2 TRANSGENIC RATS (TGR)

Objective: Comparing BP response to Ca channel opener BAY K8644 prior and after Rho kinase inhibition we demonstrated lowered calcium sensitization in spontaneously hypertensive rats (SHR), although BP response of intact SHR to Rho kinase inhibitor fasudil was enhanced (Behuliak et al. J Hypertens 31:2025,2013). The aim of the present study was to evaluate calcium sensitization in hypertensive TGR and their normotensive HanSD controls using the same methods. We also tried to elucidate the interference of baroreflex-activated sympathetic activity with BP response to fasudil. Design and Method: Blood pressure and heart rate responses to increasing doses of BAY K8644 (opener of L type voltage-dependent calcium channels) was determined in conscious heterozygous TGR rats (subjected to acute RAS and SNS blockade). BP response was recorded prior and after Rho kinase inhibition by fasudil and calcium sensitization was estimated from the difference between both BP response curves. Furthermore, BP-lowering effects of acute administration of increasing fasudil doses were measured in conscious intact rats and in rats subjected to combined blockade of RAS, SNS and NOS in which baroreflex efficiency was suppressed. Results: Using the BAY K8644-based approach we demonstrated moderately attenuated calcium sensitization in hypertensive TGR but enhanced acute BP response to fasudil in intact TGR compared to HanSD rats. This was accompanied by greater heart rate increase in HanSD than in TGR, indicating attenuated baroreflex efficiency in hypertensive TGR. If baroreflex operation was minimized by ganglionic blockade, fasudil-induced BP fall was substantially augmented in HanSD but unchanged in TGR. Conclusions: Calcium sensitization is attenuated in hypertensive TGR, this change being evident only in the absence of sympathetic nervous system which compensates BP changes elicited by acute Rho kinase inhibition better in normotensive than in hypertensive animals. Supported by grant AZV 15–25396A (MH CR)

Abstract P196: Chemerin Activates Procontractile Pathways in Isolated Arteries

In the vasculature, the adipokine chemerin is present in perivascular adipose tissue and causes arterial contraction through activation of the heptahelical receptor ChemR23. We presently test the hypothesis that ChemR23 is linked to procontractile signaling pathways in isolated arteries. The isolated, endothelium-denuded thoracic aorta of both male and female rats was used for measurement of isometric contraction and biochemical analyses. The peptide analog chemerin-9 (1 nm -3 μM) caused concentration-dependent contraction in both isolated aorta from male and female, with a maximum of ~80% of a phenylephrine (10 μM) contraction. The L type voltage dependent calcium channel inhibitors verapamil (1 μM) and nifedipine (1 μM), as well as the protein kinase C (PKC) inhibitor chelerythrine chloride (10 μM), abolished chemerin-9 induced contraction relative to vehicle-incubated tissues (male and female). Next most effective in inhibiting chemerin-9-induced contraction were the Rho Kinase inhibitors Y27632 (1 μM) and fasudil (1 μM), both effective in male and female aorta. The p38 MAPK inhibitor SB203850 (10 μM) and phosphoinositide-3-kinase inhibitor LY294002 (10 μM) were significantly less effective in reducing chemerin-9-induced contraction in both sexes, reducing maximum contraction in male and female by less than 50%. Finally, the phospholipase C inhibitor U73122 (5 μM) and Erk MAPK inhibitor PD098059 (1 μM) did not shift or reduce chemerin-9-induced contraction compared to control. Western analyses of chemerin-9-contracted tissues validated activation of phosphoinositide-3-kinase and lack of activation of the Erk MAPKs. These data are the first to associate ChemR23 with L type channel and Rho Kinase activation, and support the hypothesis that ChemR23 taps into some but not all of the well-recognized contractile pathways. This is important knowledge for understanding the mechanisms by which chemerin could support obesity-associated hypertension

Imaging Genetics Approaches to Identify Mechanisms in Severe Mental Illness

Schizophrenia, bipolar disorder, and major depressive disorders are heritable serious mental illnesses (SMI). What does this heritability mean? What are the underlying mechanisms? Large genome-wide association studies (GWAS) have identified both shared and unique genetic risk variants for these disorders; however, causal risk variants, their molecular mechanisms, and their effects on brain circuitry are mostly unknown. Targeted, possibly individualized, therapies addressing underlying mechanisms offer the promise of improved functioning and well-being for patients. The study of the effects of the CACNA1C gene on episodic memory-associated neural circuitry by Erk et al. (1) provides a noteworthy use of the imaging-genetics approach to aid in our understanding of biological mechanisms underlying SMI. Typically, the imaging-genetics approach refers to the use of structural or functional imaging data to evaluate genetic variation or more infrequently, the use of imaging data to discover genes related to disorders or symptoms. The rs1006737 A allele is a risk factor for bipolar disorder, major depressive disorder, and schizophrenia in large GWAS, making the CACNA1C gene an important therapeutic target. The rs1006737 A allele itself may not be the causal variant but may merely “tag” the true causal DNA variants. These causal variants tagged by the rs1006737 single nucleotide polymorphism (SNP) may or may not be identical among individuals, both within and across disorders. This can be resolved by DNA sequencing. Additionally, gene function is frequently modified by other factors including other genes (e.g., epistasis), methylation (epigenetics), or copy number variations or transposable element changes in DNA structure (2). Genetic variation can cause dysfunction in the CACNA1C (also called Cav1.2), which encodes the alpha-1 subunit protein of a longterm (L)-type voltage-dependent calcium channel that mediates calcium ion influx. The calcium channel consists of alpha-1, alpha-2/ delta, and beta subunits in a 1:1:1 ratio. Inactivation of the CACNA1C gene produces profound memory effects in mice. Electrophysiological, biochemical, and behavioral analyses of mice lacking the Cav1.2 gene strongly support the role for L-type voltage calciumdependent function in hippocampus-based learning and memory (3). The influx of calcium through these channels triggers hippocampus-dependent synaptic plasticity associated with memory. Interestingly, this effect on long-term potentiation appears to be N-methyl-D-aspartate-independent, in contrast to other long-term potentiation mechanisms. L-type calcium channel blockers such as nimodipine and verapamil both bind to these calcium channels and have some demonstrated efficacy in treating bipolar disorder and schizoaffective disorder. Incidentally long QT syndrome, as well as QTc (a measure of time between the beginning of the Q wave and beginning of the T wave in the heart's electrical cycle, corrected

The Interaction of Calcium Entry and Calcium Sensitization in the Control of Vascular Tone and Blood Pressure of Normotensive and Hypertensive Rats

Increased systemic vascular resistance is responsible for blood pressure (BP) elevation in most forms of human or experimental hypertension. The enhanced contractility of structurally remodeled resistance arterioles is mediated by enhanced calcium entry (through L type voltage-dependent calcium channels - L-VDCC) and/or augmented calcium sensitization (mediated by RhoA/Rho kinase pathway). It is rather difficult to evaluate separately the role of these two pathways in BP control because BP response to the blockade of either pathway is always dependent on the concomitant activity of the complementary pathway. Moreover, vasoconstrictor systems enhance the activity of both pathways, while vasodilators attenuate them. The basal fasudil-sensitive calcium sensitization determined in rats deprived of endogenous renin-angiotensin system (RAS) and sympathetic nervous system (SNS) in which calcium entry was dose-dependently increased by L-VDCC opener BAY K8644, is smaller in spontaneously hypertensive rats (SHR) than in normotensive Wistar-Kyoto (WKY) rats. In contrast, if endogenous RAS and SNS were present in intact rats, fasudil caused a greater BP fall in SHR than WKY rats. Our in vivo experiments indicated that the endogenous pressor systems (RAS and SNS) augment calcium sensitization mediated by RhoA/Rho kinase pathway, whereas the endogenous vasodilator systems (such as nitric oxide) attenuate this pathway. However, the modulation of calcium entry and calcium sensitization by nitric oxide is strain-dependent because NO deficiency significantly augments low calcium entry in WKY and low calcium sensitization in SHR. Further in vivo and in vitro experiments should clarify the interrelationships between endogenous vasoactive systems and the contribution of calcium entry and/or calcium sensitization to BP maintenance in various forms of experimental hypertension.

Genome-Wide Detection of Allele Specific Copy Number Variation Associated with Insulin Resistance in African Americans from the HyperGEN Study

African Americans have been understudied in genome wide association studies of diabetes and related traits. In the current study, we examined the joint association of single nucleotide polymorphisms (SNPs) and copy number variants (CNVs) with fasting insulin and an index of insulin resistance (HOMA-IR) in the HyperGEN study, a family based study with proband ascertainment for hypertension. This analysis is restricted to 1,040 African Americans without diabetes. We generated allele specific CNV genotypes at 872,243 autosomal loci using Birdsuite, a freely available multi-stage program. Joint tests of association for SNPs and CNVs were performed using linear mixed models adjusting for covariates and familial relationships. Our results highlight SNPs associated with fasting insulin and HOMA-IR (rs6576507 and rs8026527, 3.7*10−7≤P≤1.1*10−5) near ATPase, class V, type 10A (ATP10A), and the L Type voltage dependent calcium channel (CACNA1D, rs1401492, P≤5.2*10−6). ATP10A belongs to a family of aminophospholipid-transporting ATPases and has been associated with type 2 diabetes in mice. CACNA1D has been linked to pancreatic beta cell generation in mice. The two most significant copy variable markers (rs10277702 and rs361367; P<2.0*10−4) were in the beta variable region of the T-cell receptor gene (TCRVB). Human and mouse TCR has been shown to mimic insulin and its receptor and could contribute to insulin resistance. Our findings differ from genome wide association studies of fasting insulin and other diabetes related traits in European populations, highlighting the continued need to investigate unique genetic influences for understudied populations such as African Americans.

Characterization of voltage-dependent calcium channel blocking peptides from the venom of the tarantula Grammostola rosea

Voltage-dependent calcium channel blocking peptides were purified and sequenced from the venom of the tarantula, Grammostola rosea. cDNAs encoding the peptide sequences were cloned from the venom gland cDNA library. The electrophysiological effects of the peptides on several types of voltage-dependent calcium channels were evaluated using a Xenopus laevis oocyte expression system. A peptide contained in one of the HPLC peak fractions inhibited P/Q type voltage-dependent calcium channels (Cav2.1). The amino acid sequence of this peptide is identical to that of ω-grammotoxin SIA. A peptide from another discrete peak, which is identical to GsAFII except for one tryptophan residue in the C-terminus, inhibited L-type voltage-dependent calcium channels (Cav1.2). A novel peptide, named GTx1-15 (Accession number, AB201016), shows 76.5% sequence homology with the sodium channel blocker phrixotoxin 3, however, GTx1-15 preferentially inhibited T-type voltage-dependent calcium channels (Cav3.1). In silico secondary and tertiary structure prediction revealed that GTx1-15 and sodium channel blockers such as hainantoxin-IV, phrixotoxin 3, and ceratotoxin 2 show very similar β-strand composition, distribution of Optimal Docking Areas (continuous surface patches likely to be involved in protein–protein interactions), and surface electrostatic potential. These findings suggest that these peptide toxins evolved from common ancestors by gene duplication to maintain surface atmospheres appropriate for interaction with low-voltage-dependent ion channels.

Myoclonin1/EFHC1 disease mechanisms in JME

Summary Myoclonin1/EFHC1 mutations cause 9% of juvenile myoclonic epilepsy (JME) by impairing apoptosis in neurons/synapses where R type voltage dependent calcium channel function is altered, imbuing susceptibility to myoclonic and grand mal seizures. By disrupting mitotic spindle assembly and radial migration of neuroblasts, mutations could produce abnormal intracortical architecture upon which epileptogenesis is established. For an expanded treatment of this topic see Jasper’s Basic Mechanisms of the Epilepsies, Fourth Edition (Noebels JL, Avoli M, Rogawski MA, Olsen RW, Delgado‐Escueta AV, eds) published by Oxford University Press (available on the National Library of Medicine Bookshelf [NCBI] at http://www.ncbi.nlm.nih.gov/books).

Endothelin-1 (ET-1) Increases the Expression of Remodeling Genes in Vascular Smooth Muscle through Linked Calcium and cAMP Pathways: ROLE OF A PHOSPHOLIPASE A2(cPLA2)/CYCLOOXYGENASE-2 (COX-2)/PROSTACYCLIN RECEPTOR-DEPENDENT AUTOCRINE LOOP

Several important genes that are involved in inflammation and tissue remodeling are switched on by virtue of CRE response elements in their promoters. The upstream signaling mechanisms that inflammatory mediators use to activate cAMP response elements (CREs) are poorly understood. Endothelin (ET) is an important vasoactive mediator that plays roles in inflammation, vascular remodeling, angiogenesis, and carcinogenesis by activating 7 transmembrane G protein-coupled receptors (GPCR). Here we characterized the mechanisms ET-1 uses to regulate CRE-dependent remodeling genes in pulmonary vascular smooth muscle cells. These studies revealed activation pathways involving a cyclooxygenase-2 (COX-2)/prostacyclin receptor (IP receptor) autocrine loop and an interlinked calcium-dependent pathway. We found that ET-1 activated several CRE response genes in vascular smooth muscle cells, particularly COX-2, amphiregulin, follistatin, inhibin-β-A, and CYR61. ET-1 also activated two other genes epiregulin and HB-EGF. Amphiregulin, follistatin, and inhibin-β-A and epiregulin were activated by an autocrine loop involving cPLA2, arachidonic acid release, COX-2-dependent PGI2 synthesis, and IP receptor-linked elevation of cAMP leading to CRE transcription activation. In contrast COX-2, CYR61, and HB-EGF transcription were regulated in a calcium-dependent, COX-2 independent, manner. Observations with IP receptor antagonists and COX-2 inhibitors were confirmed with IP receptor or COX-2-specific small interfering RNAs. ET-1 increases in intracellular calcium and gene transcription were dependent upon ETa activation and calcium influx through T type voltage-dependent calcium channels. These studies give important insights into the upstream signaling mechanisms used by G protein-coupled receptor-linked mediators such as ET-1, to activate CRE response genes involved in angiogenesis, vascular remodeling, inflammation, and carcinogenesis.

The p62 antibody reveals various cytoplasmic protein aggregates in spinocerebellar ataxia Type 6

Neuronal protein aggregates are considered as pathological hallmarks of various human neurodegenerative diseases, including the so-called CAG-repeat disorders, such as spinocerebellar ataxia Type 6 (SCA6). Since the immunocytochemical findings of an initial post-mortem study using a specific antibody against the disease protein of SCA6 (i.e., pathologically altered alpha-1A subunit of the P/Q type voltage-dependent calcium channel, CACNA1A) have not been confirmed so far, the occurrence and central nervous system distribution of neuronal protein aggregates in SCA6 is still a matter of debate. Owing to the fact that the antibody against the pathologically altered CACNA1A is not commercially available, we decided to apply a recently generated p62 antibody on brain tissue from two clinically diagnosed and genetically confirmed SCA6 patients. Application of this p62 antibody revealed numerous cytoplasmic neuronal inclusions in the degenerated cerebellar dentate nucleus and inferior olive of both SCA6 patients studied, whereby a subset of these aggregates were also ubiquitin-immunopositive. In view of the known role of p62 in protein degradation as well as aggresome/sequestosome formation, the p62 aggregate formation observed in the present study suggests that SCA6 not only is associated with an impairment of the calcium channel function and an elongated polyglutamine stretch in CACNA1A, but also with a defective protein handling by the protein quality control system.

Episodic ataxia type 2

Episodic ataxia type 2 is an autosomal dominant paroxysmal cerebellar ataxia characterized by acetazolamide-responsive recurrent attacks with interictal nystagmus. This disease is caused by mutations (mainly truncating mutation) within the alpha1(A) subunit of P/Q type voltage-dependent calcium channel gene, CACNA1A. Further researches would establish the genotype/phenotype correlation and clarify the mechanism of this disorder.

Alternative splicing of exons in the alpha1 subunit of the rat testis L-type voltage-dependent calcium channel generates germ line-specific dihydropyridine binding sites.

Cell-specific isoforms of the alpha1 subunit of the L-type voltage-dependent calcium channel (VDCC) have unique pharmacological reactivities. Prior sequence analysis of nucleotide bases 3908-6077 of the VDCC alpha1 subunit expressed in rat testis differed from cardiac sequences only in a 84 base pair region corresponding to exons 31/32 encoding a putative dihydropyridine binding region. We now report that sequence analysis of bases 3048-3936 identifies a second difference between the rat testis and rat cardiac alpha1 sequence in a 60 base pair region corresponding to exons 21/22 and encoding another putative dihydropyridine binding site. Variable VDCC exons 21/22 and 31/32 and their linking introns were sequenced using genomic DNA from rat lung as template, providing evidence that the rat testis and cardiac alpha1 isoforms are products of the same gene. Reverse transcription in-situ polymerase chain reaction (PCR) with frozen sections of rat testis was carried out with primers identifying the testis-specific exon 32 of the VDCC alpha1 subunit. PCR products were confined to seminiferous tubules and were associated with the germ cell lineage from Type A spermatogonia to mature spermatozoa. Close coupling of testis alpha1 VDCC gene transcription and translation was established by in-situ immunolabelling of serial frozen sections with a monoclonal antibody (IIF7) directed against epitopes on rabbit skeletal muscle L-type VDCC alpha1. Western blot analysis of rat proteins extracted from heart, skeletal muscle, testis and spermatozoa which were reactive with the IIF7 antibody detected primarily 175-220 kDa proteins in the size range of VDCC. These data unequivocally demonstrate that an L-type VDCC is expressed in rat testis and that VDCC isoforms from rat testis and heart differ in deduced amino acid composition in and around potential binding sites for calcium channel blocking drugs such as the dihydropyridines.

Reduced levels of messenger ribonucleic acid for calcium channel, glucose transporter-2, and glucokinase are associated with alterations in insulin secretion in fasted rats.

Expression of the genes for voltage-dependent calcium channels (VDCCs), glucose transporter-2 (GLUT2), and glucokinase was studied in pancreatic islets obtained from normal rats after periods of fasting and refeeding using a competitive polymerase chain reaction procedure. A 72-h fast induced about a 3-fold decrease in the beta-cell/neuroendocrine type VDCC alpha 1-subunit and GLUT2 messenger RNA (mRNA) levels and about a 2-fold decrease in insulin and glucokinase mRNA levels compared to those in fed and refed rats. No significant differences were found in beta-actin and the cardiac-type VDCC alpha 1-subunit mRNA levels among fed, fasted, anf refed rats. We also studied insulin secretion from the isolated perfused pancreata obtained from these animals. We found an elevated threshold and decreased insulin release in response to a stepwise increase in glucose concentrations in the isolated perfused pancreata obtained from fasted rats. Fasting also resulted in a dramatic decrease in insulin secretory responses during the application of an L-type VDCC agonist, Bay K8644 (1 microM). Furthermore, fasting resulted in a significant decrease in both 45Ca2+ uptake by the isolated islets and insulin release from the islets. A strong positive correlation was observed between glucose-induced 45Ca2+ uptake and insulin output among the animals studied. On the other hand, after a 24-h refeeding, significant increases in the insulin secretory response to glucose and Bay K8644 were found, with a normalization in mRNA levels for these components. It, thus, appears that the alterations in beta-cell sensitivity to glucose that occur with fasting and refeeding are the result of complex metabolic alterations in the islet associated with reductions in expression of at least in part the beta-cell/neuroendocrine type VDCC in addition to two components of the glucose-sensing apparatus, including glucokinase and GLUT2, and the reduction in mRNA for insulin.