Sites of proteolytic processing and noncovalent association of the distal C-terminal domain of CaV1.1 channels in skeletal muscle

In skeletal muscle cells, voltage-dependent potentiation of Ca2+ channel activity requires phosphorylation by cAMP-dependent protein kinase (PKA) anchored via an A-kinase anchoring protein (AKAP15), and the most rapid sites of phosphorylation are located in the C-terminal domain. Surprisingly, the site of interaction of the complex of PKA and AKAP15 with the alpha1-subunit of Ca(V)1.1 channels lies in the distal C terminus, which is cleaved from the remainder of the channel by in vivo proteolytic processing. Here we report that the distal C terminus is noncovalently associated with the remainder of the channel via an interaction with a site in the proximal C-terminal domain when expressed as a separate protein in mammalian nonmuscle cells. Deletion mapping of the C terminus of the alpha1-subunit using the yeast two-hybrid assay revealed that a distal C-terminal peptide containing amino acids 1802-1841 specifically interacts with a region in the proximal C terminus containing amino acid residues 1556-1612. Analysis of the purified alpha1-subunit of Ca(V)1.1 channels from skeletal muscle by saturation sequencing of the intracellular peptides by tandem mass spectrometry identified the site of proteolytic processing as alanine 1664. Our results support the conclusion that a noncovalently associated complex of the alpha1-subunit truncated at A1664 with the proteolytically cleaved distal C-terminal domain, AKAP15, and PKA is the primary physiological form of Ca(V)1.1 channels in skeletal muscle cells.     

The A-kinase anchoring protein Yotiao binds and regulates adenylyl cyclase in brain

A-kinase anchoring proteins (AKAPs) influence the spatial and temporal regulation of cAMP signaling events. Anchoring of PKA in proximity to certain adenylyl cyclase (AC) isoforms is thought to enhance the phosphorylation dependent termination of cAMP synthesis. Using a combination of immunoprecipitation and enzymological approaches, we show that the plasma membrane targeted anchoring protein AKAP9/Yotiao displays unique specificity for interaction and the regulation of a variety of AC isoforms. Yotiao inhibits AC 2 and 3, but has no effect on AC 1 or 9, serving purely as a scaffold for these latter isoforms. Thus, Yotiao represents an inhibitor of AC2. The N terminus of AC2 (AC2-NT), which binds directly to amino acids 808–957 of Yotiao, mediates this interaction. Additionally, AC2-NT and Yotiao (808–957) are able to effectively inhibit the association of AC2 with Yotiao and, thus, reverse the inhibition of AC2 by Yotiao in membranes. Finally, disruption of Yotiao–AC interactions gives rise to a 40% increase in brain AC activity, indicating that this anchoring protein functions to directly regulate cAMP production in the brain.     

Mechanisms underlying the insulinostatic effect of peptide YY in mouse pancreatic islets

Summary Peptide YY is an insulinostatic peptide which is released into the circulation from the intestinal mucosa upon food intake. Peptide YY is also co-stored with glucagon in the secretory granules of the pancreatic alpha cells. We examined the mechanisms underlying the insulinostatic effect of peptide YY in isolated mouse pancreatic islets. We found that peptide YY (0.1 nmol/l-1 mol/l) inhibited glucose (11.1 mmol/l)-stimulated insulin secretion from incubated isolated islets, with a maximal inhibition of approximately 70% observed at a dose of 1 nmol/ 1 (p<0.001). Also in perifused islets the peptide (1 nmol/l) inhibited insulin secretion in response to 11.1 mmol/l glucose (p<0.001). Furthermore, peptide YY inhibited glucose-stimulated cyclic AMP formation (by 67%, p<0.05), and insulin secretion stimulated by dibutyryl cyclic AMP (p<0.01). In contrast, the peptide was without effect both on the cytoplasmic Ca²⁺ concentration in dispersed mouse islet-cell suspensions as measured by the FURA 2-AM technique, and on insulin release in isolated islets, when stimulated by the protein kinase C-activator 12-O-tetradecanoyl phorbol 13-acetate. Finally, in pre-labelled perifused islets, peptide YY caused a small and transient increase in the ⁸⁶Rb⁺ efflux (p<0.001), but only in the absence of extracellular Ca²⁺. We conclude that peptide YY inhibits glucose-stimulated insulin secretion from isolated mouse islets by inhibiting two different steps in the cyclic AMP cascade, that is, both the accumulation and the action of the cyclic nucleotide. In contrast, the data suggest that protein kinase C, K⁺ channels, the cytoplasmic Ca²⁺ concentration or other processes directly regulating the exocytosis are not involved in the signal transduction underlying peptide YY-induced inhibition of insulin secretion.Abbreviations PYY Peptide YY - TPA 12-O-tetradecanoylphorbol 13-acetate     

Role of protein kinase a in human hepatocyte DNA synthesis

The cellular mechanisms associated with the replicative response of hepatocytes to growth factor stimulation is incompletely understood. Murine hepatocyte DNA synthesis is altered by cyclic AMP, suggesting that protein kinase A is involved in the cellular mechanisms associated with liver growth. The purpose of this study was to evaluate the role of protein kinase A in human hepatocyte DNA synthesis. Human hepatocytes were isolated and maintained in primary culture on rat tail collagen. DNA synthesis was evaluated by determining [³H]thymidine incorporation. Human hepatocytes between 24 and 96 hr following harvest increased DNA synthesis in response to epidermal growth factor but not in response to glucagon, a stimulant of adenyl cyclase, or dibutyryl cyclic AMP. Mitogen-stimulated DNA synthesis was decreased by dibutyryl cyclic AMP. Cyclic AMP isomers that block or stimulate the effect of cyclic AMP on protein kinase A did not significantly alter resting or mitogen-stimulated human hepatocyte DNA synthesis. The results suggest that increased protein kinase A activity does not produce human hepatocyte replicative DNA synthesis.Supported in part by USPHS DK27695.     

Calcium channel blockers in vivo inhibit serotonin N-acetyltransferase (NAT) activity in chicken retina stimulated by darkness and not by agents elevating intracellular cyclic AMP level

The molecular mechanism underlying the role of calcium influx in the regulation of retinal serotonin N-acetyltransferase (NAT) activity was studied in vivo in chickens. Systemic administration of organic antagonists of voltage-sensitive calcium channels (VSCC), i.e., nimodipine and nifedipine, resulted in a marked suppression of the nocturnal increase of NAT activity in chicken retina. In contrast, NAT activity stimulated by nonhydrolysable analogs of cyclic AMP (dibutyryl-cyclic AMP and 8-bromo-cyclic AMP), forskolin, a direct activator of adenylate cyclase, and by phosphodiesterase inhibitors (aminophylline and 3-isobutyl-1-methylxanthine), was not significantly affected by various tested VSCC antagonists. The inhibitory effect of nimodipine on the dark-dependent increase in NAT activity of chicken retina was abolished by Bay K 8644, a selective VSCC agonist. The results presented in this paper indicate an important role of calcium influx through L-type of VSCC in the induction of NAT activity in chicken retina, and suggest that a requirement of calcium ions in the process of NAT induction in the retina may be primarily at the level of cyclic AMP production.     

The effect of calcium on the potentiation of LH-stimulated steroidogenesis and inhibition of LH-stimulated cyclic AMP production by LHRH agonist (ICI 118630) in rat Leydig cells

A luteinizing hormone-releasing hormone (LHRH) agonist (ICI 118630) potentiated the effects of luteinizing hormone (LH) and dibutyryl cyclic AMP on steroidogenesis during 4 h incubations with rat Leydig cells. LH-stimulated cyclic AMP levels were decreased by the addition of the LHRH agonist. The potentiation of the LH-increased steroidogenesis was dependent on Ca2+; maximum effects required at least 2.5 mM Ca2+ in the incubation medium. The calcium ionophore A23187 negated the potentiation in a dose-dependent manner (ED50 = 0.2-0.3 microM), but had no effect on LH-induced steroidogenesis, despite a 90% decrease in cyclic AMP production. The latter decrease was found to be dependent on the Ca2+ concentration. In the presence of the phosphodiesterase inhibitor methylisobutylxanthine (MIX), the ionophore A23187 induced a dose-dependent decrease in both LH and LH plus LHRH agonist-stimulated steroidogenesis and cyclic AMP production. The results obtained indicate that calcium, rather than cyclic AMP, is the mediator of the potentiating effects of LHRH agonist on LH-increased steroidogenesis in rat Leydig cells. The marked inhibition of the synergism in the presence of calcium ionophore A23187 suggests that Leydig cell calcium homeostasis must be intact for LHRH agonist action to occur. LHRH agonist causes a Ca2+-dependent decrease in LH-stimulated cyclic AMP production.     

Roles for both cyclic GMP and cyclic AMP in the inhibition of collagen-induced platelet aggregation by nitroprusside

In studies on human platelets, nitroprusside (NP) alone at 1-10 micromol/l increased platelet cyclic AMP (cAMP) by 40-70%, whereas increases in cyclic GMP (cGMP) were much larger in percentage though not in concentration terms. Collagen enhanced these increases in cAMP up to fourfold, without affecting cGMP. This effect was partly prevented by indomethacin or aspirin, indicating that platelet cyclo-oxygenase products acted synergistically with NP to increase cAMP. ADP released from the platelets by collagen tended to restrict this cAMP accumulation. Addition of 2',5'-dideoxyadenosine (DDA), an inhibitor of adenylyl cyclase, decreased both the inhibition of collagen-induced platelet aggregation by NP and the associated accumulation of cAMP without affecting cGMP, indicating that cAMP mediates part of the inhibitory effect of NP. Unlike DDA, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of guanylyl cyclase, blocked all increases in both cGMP and cAMP caused by NP, as well as the inhibition of platelet aggregation, suggesting that cAMP accumulation was secondary to that of cGMP. Human platelet cGMP-dependent protein kinase (PKG) coelectrophoresed with the purified bovine type Ibeta isoenzyme. An inhibitor of this enzyme (Rp)-beta-phenyl-1,N2-etheno-8-bromoguanosine 3',5'-cyclic-monophosphorothioate, diminished the inhibition of collagen-induced platelet aggregation by NP, but had little additional effect when DDA was present. This showed that both PKG and cAMP participate in the inhibition of collagen-induced platelet aggregation by NP. Moreover, selective activators of PKG and cAMP-dependent protein kinases had supra-additive inhibitory effects, suggesting that an optimal inhibitory effect of NP requires simultaneous activation of both enzymes.     

Convergent regulation of skeletal muscle Ca2+ channels by dystrophin, the actin cytoskeleton, and cAMP-dependent protein kinase

The skeletal muscle L-type Ca2+ channel (Ca(V)1.1), which is responsible for initiating muscle contraction, is regulated by phosphorylation by cAMP-dependent protein kinase (PKA) in a voltage-dependent manner that requires direct physical association between the channel and the kinase mediated through A-kinase anchoring proteins (AKAPs). The role of the actin cytoskeleton in channel regulation was investigated in skeletal myocytes cultured from wild-type mice, mdx mice that lack the cytoskeletal linkage protein dystrophin, and a skeletal muscle cell line, 129 CB3. Voltage dependence of channel activation was shifted positively, and potentiation was greatly diminished in mdx myocytes and in 129 CB3 cells treated with the microfilament stabilizer phalloidin. Voltage-dependent potentiation by strong depolarizing prepulses was reduced in mdx myocytes but could be restored by positively shifting the stimulus potentials to compensate for the positive shift in the voltage dependence of gating. Inclusion of PKA in the pipette caused a negative shift in the voltage dependence of activation and restored voltage-dependent potentiation in mdx myocytes. These results show that skeletal muscle Ca2+ channel activity and voltage-dependent potentiation are controlled by PKA and microfilaments in a convergent manner. Regulation of Ca2+ channel activity by hormones and neurotransmitters that use the PKA signal transduction pathway may interact in a critical way with the cytoskeleton and may be impaired by deletion of dystrophin, contributing to abnormal regulation of intracellular calcium concentrations in dystrophic muscle.     

Topology of the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum (RyR1)

To define the topology of the skeletal muscle ryanodine receptor (RyR1), enhanced GFP (EGFP) was fused in-frame to the C terminus of RyR1, replacing a series of C-terminal deletions that started near the beginning or the end of predicted transmembrane helices M1-M10. The constructs were expressed in HEK-293 (human embryonic kidney cell line 293) or mouse embryonic fibroblast (MEF) cells, and confocal microscopy of intact and saponin-permeabilized cells was used to determine the subcellular location of the truncated fusion proteins. The fusion protein truncated after M3 exhibited uniform cytoplasmic fluorescence, which was lost after permeabilization, indicating that proposed M', M", M1, M2, and M3 sequences are not membrane-associated. The fusion protein truncated at the end of the M4-M5 loop and containing M4 was membrane-associated. All longer truncated fusion proteins were also associated with intracellular membranes. Mapping by protease digestion and extraction of isolated microsomes demonstrated that EGFP positioned after either M5, the N-terminal half of M7 (M7a), or M8 was located in the lumen, and that EGFP positioned after either M4, M6, the C-terminal half of M7 (M7b), or M10 was located in the cytoplasm. These results indicate that RyR1 contains eight transmembrane helices, organized as four hairpin loops. The first hairpin is likely to be made up of M4a-M4b. However, it could be made up from M3-M4, which might form a hairpin loop even though M3 alone is not membrane-associated. The other three hairpin loops are formed from M5-M6, M7a-M7b, and M8-M10. M9 is not a transmembrane helix, but it might form a selectivity filter between M8 and M10.     

Molecular mechanisms and therapeutics of the deficit in specific force in ageing skeletal muscle

The age-related impairment in muscle force is only partially explained by the loss of muscle mass. The loss both in specific and absolute forces contributes to the muscle weakness measured in the elderly and in animal models of ageing. Successful interventions aimed at preventing age-associated functional deficits will require a better insight into the mechanisms underlying the decline in muscle-specific force. The present review article is focused on recent evidence supporting excitation-contraction uncoupling as a key factor underlying fast and slow muscle fiber impairment with ageing. The molecular, functional and structural factors supporting this theory and counteracting measures such as insulin-like growth factor 1 transgenic overexpression are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Stoichiometric requirements for trapping and gating of Ca2+ release-activated Ca2+ (CRAC) channels by stromal interaction molecule 1 (STIM1)

Store-operated Ca(2+) entry depends critically on physical interactions of the endoplasmic reticulum (ER) Ca(2+) sensor stromal interaction molecule 1 (STIM1) and the Ca(2+) release-activated Ca(2+) (CRAC) channel protein Orai1. Recent studies support a diffusion-trap mechanism in which ER Ca(2+) depletion causes STIM1 to accumulate at ER-plasma membrane (PM) junctions, where it binds to Orai1, trapping and activating mobile CRAC channels in the overlying PM. To determine the stoichiometric requirements for CRAC channel trapping and activation, we expressed mCherry-STIM1 and Orai1-GFP at varying ratios in HEK cells and quantified CRAC current (I(CRAC)) activation and the STIM1:Orai1 ratio at ER-PM junctions after store depletion. By competing for a limited amount of STIM1, high levels of Orai1 reduced the junctional STIM1:Orai1 ratio to a lower limit of 0.3-0.6, indicating that binding of one to two STIM1s is sufficient to immobilize the tetrameric CRAC channel at ER-PM junctions. In cells expressing a constant amount of STIM1, CRAC current was a highly nonlinear bell-shaped function of Orai1 expression and the minimum stoichiometry for channel trapping failed to evoke significant activation. Peak current occurred at a ratio of ～2 STIM1:Orai1, suggesting that maximal CRAC channel activity requires binding of eight STIM1s to each channel. Further increases in Orai1 caused channel activity and fast Ca(2+)-dependent inactivation to decline in parallel. The data are well described by a model in which STIM1 binds to Orai1 with negative cooperativity and channels open with positive cooperativity as a result of stabilization of the open state by STIM1.     

Dependence of prolactin release on coupling between Ca(2+) mobilization and voltage-gated Ca(2+) influx pathways in rat lactotrophs

Two Ca²⁺-mobilizing receptors expressed in lactotrophs, endothelin-A (ET_A) and thyrotropin-releasing hormone (TRH), induce a rapid Ca²⁺ release from intracellular stores and prolactin (PRL) secretion but differ in their actions during the sustained stimulation; TRH facilitates and ET-1 inhibits voltage-gated calcium influx (VGCI) and PRL secretion. In pertussis toxin (PTX)-treated cells, ET-1-induced inhibition of VGCI was abolished and the pattern of Ca²⁺ signaling was highly comparable with that observed in TRH-stimulated cells. The addition of Cs⁺, a relatively specific blocker of inward rectifier K⁺ channels, mimicked the effect of PTX on the pattern of ET-1-induced sustained Ca²⁺ signaling, but only in about 50% of cells, and did not affect agonist-induced inhibition of PRL secretion. Extracellular Cs⁺ was also ineffective in altering the TRH-induced facilitation of VGCI and PRL secretion. Furthermore, apamin and paxilline, specific blockers of Ca²⁺-activated SK-and BK-type K⁺ channels, respectively; E-4031, a blocker of ether a-go-go K⁺ channel; and linopirdine, a blocker of M-type K⁺ channel, did not affect the agonist-specific patterns of calcium signaling and PRL secretion. These results suggest that ET-1 inhibits VGCI through activation of Cs⁺-sensitive channels, presumably the G_i/o-controlled inward rectifier K⁺ channels, and that this agonist also inhibits PRL release, but downstream of Ca²⁺ influx. Further studies are required to identify the mechanism of sustained TRH-induced facilitation of VGCI and PRL secretion.     

胰岛素抵抗大鼠骨骼肌中蛋白激酶B表达及葡萄糖转运蛋白4转位的改变

目的研究高脂饮食喂养的胰岛素抵抗(IR)大鼠骨骼肌中蛋白激酶B(PKB)表达和葡萄糖转运蛋白4(GluT4)转位的改变及饮食治疗、葛根素、罗格列酮干预的影响。方法将雄性SD大鼠50只随机分为正常饮食(A)组和高脂饮食(B)组,2个月后再将B组大鼠随机分为高脂饮食(C)组、正常饮食干预(D)组、葛根素干预(E)组和罗格列酮干预(F)组。干预1个月后检测骨骼肌中PKB的表达及转位至质膜的GluT4含量。结果C组大鼠产生了明显的IR,骨骼肌中PKB的表达较A组显著降低(P<0.01),转位到质膜上的GluT4含量显著减低(P<0.01);D、E、F组大鼠IR明显改善,骨骼肌中PKB的表达较C组大鼠显著增加(P<0.01),GluT4含量较C组大鼠显著升高(P<0.01)。结论高脂饮食喂养的SD大鼠骨骼肌产生明显的IR,骨骼肌中Ins诱导的PKB表达降低,Ins刺激的GluT4向质膜的转位减少。饮食治疗及葛根素、罗格列酮干预能增加骨骼肌中Ins刺激的PKB表达及GluT4向质膜的转位。     

利拉鲁肽对小鼠骨骼肌细胞葡萄糖转运子4转位的作用

目的 探讨胰高血糖素样肽1 （GLP-1）类似物利拉鲁肽对小鼠骨骼肌细胞葡萄糖转运子4（GLUT4）转位的作用及可能机制.方法 在过表达带有HA表位GLUT4的小鼠骨骼肌细胞株C2C12（C2C12-GLUT4H）,分为正常对照组、胰岛素组（100 nmol/L）、利拉鲁肽1组（100 nmol/L）、利拉鲁肽2组（1 000 nmol/L）、5-氨基咪唑-4-甲酰1-β-D呋喃糖苷（AICAR）（腺苷酸活化蛋白激酶激动剂）组（2 mmol/L）.通过ELISA法测定细胞膜上C2C12-GLUT4HA,检测各组对C2C12-GLUT4HA细胞GLUT4转位的作用.应用Western blotting检测介导GLUT4转位的信号分子蛋白激酶B（AKT）、腺苷酸活化蛋白激酶（AMPK）磷酸化水平以及GLUT4蛋白表达水平.采用Student＇s t检验或单因素方差分析进行统计分析.结果 利拉鲁肽组的GLUT4转位较对照组相比明显上升[分别是对照组的（1.53±0.28）倍、（1.41±0.41）倍,F=13.4798,P＜0.05];利拉鲁肽刺激组与对照组相比能够使pAMPK水平升高[分别是对照组的（1.79±0.31）倍、（1.54±0.18）倍,F=20.0999,P＜0.05],而对pAKT无明显影响（P＞0.05）.结论 利拉鲁肽可通过激活AMPK从而促进小鼠骨骼肌细胞GLUT4转位的增加.     

Effect of testosterone on lipolysis in human pre-adipocytes from different fat depots

Aim/hypothesis Regional differences in lipolysis, with higher lipolytic activity in visceral than subcutaneous fat, are important for the development of insulin resistance and might be influenced by testosterone.Methods We studied testosterone-regulated lipolysis and protein expression (by western blot) in fully differentiated pre-adipocytes from visceral (omental) and abdominal subcutaneous adipose tissue from 52 human subjects. These cells were isolated and cultured in a serum-free medium.Results Testosterone caused a specific, time- and concentration-dependent 50% reduction of catecholamine-stimulated lipolysis in the subcutaneous depot. Half of the maximum effect occurred at 10 nmol/l. The inhibitory effect was due to the inability of -adrenoceptors and cyclic AMP to stimulate the protein kinase A, hormone-sensitive lipase complex. Testosterone caused a depot-specific 50% reduction of the protein expression of hormone-sensitive lipase and ₂-adrenoceptors in differentiated subcutaneous pre-adipocytes, but no change in ₁-adrenoceptors, protein kinase A subunits or perilipin expression. In contrast, testosterone had no effect on lipolysis or protein expression in the visceral depot. However, testosterone receptors were present in both depots, and the hormone inhibited adipocyte leptin secretion. Similar effects on lipolysis were observed with dihydrotestosterone.Conclusions/interpretation Testosterone in physiological concentrations causes a depot-specific reduction of catecholamine-stimulated lipolysis in subcutaneous fat cells, probably due to reduced protein expression of ₂-adrenoceptors and hormone-sensitive lipase. This could be an important pathogenic factor underlying regional differences in lipolysis and development of insulin resistance and hyperandrogenic polycystic ovary syndrome.Abbreviations PCOS polycystic ovary syndrome - GPDH glycerol-3-phosphate dehydrogenase - AR adrenergic receptor - HSL hormone-sensitive lipase - PKA protein kinase A - OD optical density - dcAMP dibutyryl cyclic AMP     

Rem蛋白表达对胚胎心室肌细胞Ca2+通道功能的影响

目的:研究RGK家族中Rem微小蛋白对胚胎小白鼠心室肌细胞Ca~(2 )通道功能的影响。方法:用含有相同浓度正常的Rem(Ad-RemWT)和反转的Rem蛋白(Ad-CTRrem)的两组重组腺病毒(1012/ml)分别感染培养的怀孕18d胚胎小白鼠(ED18)心室肌细胞。12h后,用整个细胞的电压嵌技术来记录ED18心室肌细胞膜L-型Ca~(2 )电流,细胞质Ca~(2 )转运用荧光光度测定法测定。结果:Rem可以很好地在ED18心室肌细胞中表达。在整个细胞的电压嵌技术下,对照组(Ad-CTRrem)能记录到L-型Ca~(2 )电流的细胞为83·3%,实验组(Ad-Rem-WT)为6·6%。Rem有效阻断了ED18心室肌细胞膜Ca~(2 )通道电流。通过荧光光度测定法,测定Rem显著减低了ED18心室肌细胞质Ca~(2 )的转运。结论:外源的RGK家族中Rem蛋白的表达,通过与心室肌细胞Ca~(2 )通道蛋白的β亚单位(Cavβ)相结合改变了Ca~(2 )通道功能,限制了Ca~(2 )的内流。     

Additive effects of norepinephrine and cyclic AMP on the activation of the protein kinase from adipose tissue.

In this work the kinetics of activation of the cyclic AMP-dependent protein kinase by several catecholamines and ACTH, have been studied in rat epididymal fat pads and isolated fat cells. The method of Soderling and co-workers which permits the measurement of the state of activation of the protein kinase after hormonal stimulation in adipose tissue, has been used. Kinetics experiments where norepinephrine was used showed that the results obtained with isolated cells conform to the models of Sutherland and Brostrom and co-workers. Wtih intact tissue, norepinephrine not only stimulates the protein kinase activity measured without exogenous cyclic AMP but also the total activity measured in the presence of cyclic AMP (5 X 10(-6) M); thus the effect of norepinephrine, obtained during incubation of the tissue, and that of cyclic AMP, added to the soluble fraction after incubation, were additive. This effect seems to be of the beta type because it is blocked completely by propranolol. A weak, additive but significant effect was also obtained with epinephrine and isoproterenol but not with ACTH. Neither cyclic GMP nor cyclic IMP seems implicated in this effect. It was shown that stroma vascular cells which are present in the fat pads are not involved. These results suggest that the effects of norepinephrine on the protein kinase of the fat pads cannot be completely explained by the model of Brostrom and colleagues.     

Increased phosphorylation of the neuronal L-type Ca(2+) channel Ca(v)1.2 during aging

An increase in Ca2+ influx through L-type Ca2+ channels is thought to contribute to neuronal dysfunctions that underlie senile symptoms and Alzheimer's disease. The molecular basis of the age-dependent up-regulation in neuronal L-type Ca2+ channel activity is largely unknown. We show that phosphorylation of the L-type channel Cav1.2 by cAMP-dependent protein kinase is increased >2-fold in the hippocampus of aged rats. The hippocampus is critical for learning and is one of the first brain regions to be affected in Alzheimer's disease. Phosphorylation of Cav1.2 by cAMP-dependent protein kinase strongly enhances its activity. Therefore, increased Cav1.2 phosphorylation may account for a substantial portion of the age-related rise in neuronal Ca2+ influx and its neuropathological consequences.