Transport of 3-O-methyl-d-glucose into mammalian pancreatic β-cells

Summary The transport and oxidation of 3-O-methyl-d-(U-¹⁴C)glucose was studied in microdissected pancreatic islets of obese-hyperglycemic mice. There was no significant production of¹⁴CO₂ during incubation for 2 h. A comparison with the uptake of sucrose and mannitol indicated that 3-O-methyl-d-glucose was uniformly distributed across the -cell plasma membrane. Externald-glucose inhibited the entry of 3-O-methyl-d-glucose and caused a significant net loss of 3-O-methyl-d-glucose from islets equilibrated with this compound. The transport of 3-O-methyl-D-glucose was also markedly reduced in the presence of phlorizin or phloretin, whereasd-mannoheptulose ord-glucosamine exerted a slight inhibition. The results support our hypothesis that the transport ofd-glucose into the pancreatic -cells is carriermediated, and indicate that 3-O-methyl-d-glucose is a non-metabolizable substrate for this carrier in the pancreatic islets. Since in contrast tod-glucose 3-O-methyl-d-glucose does not stimulte insulin release from the type of islets used, the secretagoric recognition system ford-glucose is probably not identical with the membrane transport system.     

Permeability to Ca2+ of the acetylcholine receptor channel of denervated mouse muscles in the presence of Na+ and of some other cations

Calcium uptake caused by exposure to azelainylcholine and the additional membrane slope conductance caused by the same agonist were compared in partially depolarized mouse soleus muscles denervated for 3–5 days. Ca uptake was estimated from the amount of⁴⁵Ca retained after a 2 min exposure to the tracer (1 min in the presence of azelainylcholine) and a subsequent 17 min period of tracer washout. The amount taken up in the presence of Na⁺ was 0.152 m mole/kg fresh muscle. The uptake was by about 60% higher when Na⁺ was replaced by N-methyl-d-glucamine. For 10 other monovalent cations Ca uptake was less than with Na. Ca uptake was not related to the molecular weight, size or structure of the cation. The slope conductance in the presence of 10 M azelainylcholine was 4 S and it was 21% of that value when Na⁺ was replaced by N-methyl-d-glucamine, i.e. conductance was decreased when Ca uptake was increased. This discrepancy points to a major difference in the way cations such as Ca²⁺ and K⁺ pass the receptor channel.     

The stimulus-secretion coupling of amino acid-induced insulin release

l-Glutamine enhances insulin release evoked byl-leucine in isolated rat pancreatic islets. The enhancing action ofl-glutamine, which is a rapid but steadily increasing and not rapidly reversible phenomenon, is not attributable to any major change in either K⁺ or Ca²⁺ outflow from the islet cells. It coincides with an apparent increase in Ca²⁺ inflow rate and, hence, with Ca accumulation in the islets. The initial ionic response tol-leucine is not qualitatively altered by the presence ofl-glutamine. In their combined capacity to stimulate⁴⁵Ca net uptake in the islets,l-glutamine can be replaced byl-asparagine but not byl-glutamate, whereasl-leucine can be replaced byl-norvaline orl-isoleucine, but not byl-valine, glycine orl-lysine. Such a specificity is identical to that characterizing the effect of these various amino acids upon insulin release. It is postulated that the release of insulin evoked by the combination ofl-leucine andl-glutamine involves essentially the same remodelling of ionic fluxes as that evoked by other nutrient secretagogues with, however, an unusual time course for the functional response tol-glutamine.     

Mechanism of the enhancing effect of sorbitol on ileal Ca uptake in rat enterocytes

The effect of sorbitol on Ca uptake by isolated ileal epithelial cells was investigated. Intestinal cells were isolated from rat ileum by mechanical vibration.⁴⁵Ca uptake was approximately 2 times higher in cells exposed to 200 mM sorbitol ofd-alanine than in control cells. This enhancing effect of sorbitol on percentage Ca uptake decreased with increasing Ca concentrations in the incubation medium suggesting an effect on Ca entry velocity. The addition of 10 M nifedipine or 200 M verapamil to the incubation medium was devoid of any effect on Ca uptake in ileal cells, whereas 100 M trifluoperazine or chlorpromazine abolished the stimulatory effect of sorbitol. Finally, the effect of sorbitol on isolated cells was independent of a measurable change of cellular ATP content. In conclusion, the stimulatory effect of sorbitol on ileal Ca uptake is probably exerted through mechanisms other than an increase in intracellular ATP concentration. Sorbitol may enhance enterocyte Ca transport via a direct interaction with calmodulin and/or the Ca pump. It may also exert its effect through an inhibition of the basolateral Na Ca exchanger.     

TTX-sensitive Na+ channels and Ca2+ channels of the Land N-type underlie the inward current in acutely dispersed coeliac-mesenteric ganglia neurons of adult rats

Inward membrane currents of sympathetic neurons acutely dispersed from coeliac-superior mesenteric ganglia (C-SMG) of adult rats were characterized using the whole-cell variant of the patch-clamp technique. Current-clamp studies indicated that C-SMG neurons retained electrical properties similar to intact ganglia. Voltage-clamp studies designed to isolate Na⁺ currents revealed that tetrodotoxin (TTX, 1 M) completely inhibited the large transient inward current. Half activation potential (V _h) and slope factor (K) were –26.8 mV and 6.1 mV, respectively. Inactivation parameters for V _h and K were –65 mV and 8.2 mV, respectively. Voltage-clamp studies also revealed a high-voltage-activated sustained inward Ca²⁺ current which was blocked by the removal of external Ca²⁺ or the presence of Cd²⁺ (0.1 mM). The dihydropyridine agonist, (+)202–791 (1 M), caused a small increase (20%) in the amplitude of the Ca²⁺ current at more negative potentials and markedly prolonged the tail currents. -Conotoxin GIVA (, CgTX, 15 M) caused a 66% inhibition of the high-voltage-activated Ca²⁺ current amplitude. Norepinephrine (1 M) caused a 49% reduction in the peak Ca²⁺ current. This study is the first demonstration that dispersed C-SMG neurons from adult rats retain electrical characteristics similar to intact ganglia. A TTX-sensitive Na⁺ current as well as a high voltage-activated sustained Ca²⁺ current underlie the inward current in C-SMG neurons. The macroscopic Ca²⁺ current is composed of a small dihydropyridinesensitive (L-type current) and a large -CgTx-sensitive (N-type current) component. Thus, acutely dispersed CSMG neurons are suitable for examining the biophysical properties and modulation of membrane currents of adult prevertebral sympathetic neurons in normal and diseased states.     

ATP-induced intracellular Ca2+ signals in isolated human insulin-secreting cells

Using isolated -cells from human islets of Langerhans we have demonstrated that purinergic receptor agonists are functionally coupled to rises in the intracellular calcium ion concentration ([Ca²⁺]_i). The effects of ATP, ADP and AMP have been examined over a range of concentrations, 0.5 to 500M. The actions of ATP were more potent than those of either ADP or AMP suggesting that a P₂-type of purinergic receptor operates in these cells. Responses to ATP were concentration-related, but exhibited marked desensitisation at high concentrations (>100M). Purinergic receptor agonists elevate [Ca²⁺]_i by mechanisms that involve both Ca²⁺ influx and Ca²⁺ mobilisation from intracellular stores. The physiological significance of our data has been discussed, and related to previous studies carried out upon rodent and clonal insulin-secreting cells.     

Bursting electrical activity in pancreatic β-cells: evidence that the channel underlying the burst is sensitive to Ca2+ influx through L-type Ca2+ channels

In glucose-stimulated pancreatic -cells, the membrane potential alternates between a hyperpolarized silent phase and a depolarized phase with Ca²⁺ action potentials. The molecular and ionic mechanisms underlying these bursts of electrical activity remain unknown. We have observed that 10.2–12.8 mM Ca²⁺, 1 M Bay K 8644 and 2 mM tetraethylammonium (TEA) trigger bursts of electrical activity and oscillations of intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in the presence of 100 M tolbutamide. The [Ca²⁺]_i was monitored from single islets of Langerhans using fura-2 microfluorescence techniques. Both the high-Ca²⁺ and Bay-K-8644 evoked [Ca²⁺]_i oscillations overshot the [Ca²⁺]_i recorded in tolbutamide. Nifedipine (10–20 M) caused an immediate membrane hyperpolarization, which was followed by a slow depolarization to a level close to the burst active phase potential. The latter depolarization was accompanied by suppression of spiking activity. Exposure to high Ca²⁺ in the presence of nifedipine caused a steady depolarization of approximately 8 mV. Ionomycin (10 M) caused membrane hyperpolarization in the presence of 7.7 mM Ca²⁺, which was not abolished by nifedipine. Charybdotoxin (CTX, 40–80 nM), TEA (2 mM) and quinine (200 M) did not suppress the high-Ca²⁺-evoked bursts. It is concluded that: (1) the channel underlying the burst is sensitive to [Ca²⁺]_i rises mediated by Ca²⁺ influx through L-type Ca²⁺ channels, (2) both the ATP-dependent K⁺ channel and the CTX and TEA-sensitive Ca²⁺-dependent K⁺ channel are highly unlikely to provide the pacemaker current underlying the burst. We propose that the burst is mediated by a distinct Ca²⁺-dependent K⁺ channel and/or by [Ca²⁺]_idependent slow processes of inactivation of Ca²⁺ currents.     

Forskolin-induced block of delayed rectifying K+ channels in pancreaticβ-cells is not mediated by cAMP

K⁺ channels in the membrane of murine pancreatic -cells were studied using the patch-clamp technique. The delayed outward current was activated in whole-cell experiments by depolarizing voltage pulses to potentials between –30 mV and 0 mV. Forskolin blocked the current rapidly (<5 s) and reversibly with 50% inhibition at 13 M. The inhibition did not depend on a stimulation of the adenylate cyclase since it occurred even in presence of 1 mM cAMP in the pipette solution which replaced the cytoplasm. Membrane permeant cAMP analogues and phosphodiesterase inhibitors did not influence the delayed outward current. In experiments on outside-out patches forskolin (100 M) shortened the openings of a channel of about 10 pS conductance at 0 mV and a time course of activation and inactivation similar to the whole-cell current. Another smaller, slowly activating channel and the Ca²⁺- and ATP-dependent K⁺ channels were influenced only weakly or not at all. It is therefore concluded that the 10-pS channel generates most of the delayed outward K⁺ current in murine pancreatic -cells. The Ca²⁺-independent part of the delayed outward current in bovine adrenal chromaffin cells was also blocked by forskolin (100 M).     

Multiple calcium channel subtypes in isolated rat chromaffin cells

By using the whole-cell configuration of the patch-clamp technique we have investigated the pharmacological properties of Ca²⁺ channels in short-term cultured rat chromaffin cells. In cells held at a membrane potential of –80 mV, using 10 mM Ba²⁺ as the charge carrier, only high-voltage-activated (HVA) Ca²⁺ channels were found. Ba²⁺ currents (I _Ba) snowed variable sensitivity to dihydropyridine (DHP) Ca²⁺ channel agonists and antagonists. Furnidipine, a novel DHP antagonist, reversibly blocked the current amplitude by 22% and 48%, at 1 M and 10 M respectively, during short (15–50 ms) depolarizing pulses to 0 mV. The L-type Ca²⁺ channel agonist Bay K 8644 (1 M) caused a variable potentiation of HVA currents that could be better appreciated at low rather than at high depolarizing steps. Increase of I _Ba was accompanied by a 20-mV shift in the activation curves for Ca²⁺ channels towards more hyperpolarizing potentials. Application of the conus toxin -conotoxin GVIA (GVIA; 1 M) blocked 31% of I _Ba; blockade was irreversible upon removal of the toxin from the extracellular medium, -Agatoxin IVA (IVA; 100 nM) produced a 15% blockade of I _Ba. -Conotoxin MVIIC (MVIIC; 5 M) produced a 36% blockade of I _Ba; such blockade seems to be related to both GVIA-sensitive (N-type) and GVIA-resistant Ca²⁺ channels. The sequential addition of supramaximal concentrations of furnidipine (10 M), GVIA (1 M), IVA (100 nM) and MVIIC (3 M) produced partial inhibition of I _Ba, which were additive. Our data suggest that the whole cell I _Ba in rat chromaffin cells exhibits at least four components. About 50% of I _Ba is carried by L-type Ca²⁺ channels, 30% by N-type Ca²⁺channels and 15% by P-type Ca²⁺ channels. These figures are close to those found in cat chromaffin cells. However, they differ considerably from those found in bovine chromaffin cells where P-like Ca²⁺channels account for 45% of the current, N-type carry 35% and L-type Ca²⁺ channels are responsible for only 20–25% of the current. These drastic differences might have profound physiological implications for the relative contribution of each channel subtype to the regulation of catecholamine release in different animal species.     

The dual effects of ouabain on45Ca2+ transport and contractility in adult rat ventricular myocytes

We used isolated ventricular myocytes to study⁴⁵Ca²⁺ transport in the presence of three concentrations of ouabain (10 nM, 1 M, and 100 M) in Tyrode solution containing 1 mM CaCl₂. The cells were quiescent and during⁴⁵Ca²⁺ uptake and⁴⁵Ca²⁺ efflux experiments 10 nM ouabain decreased Ca²⁺ content, 1 M, didn't change it appreciably, and 100 M increased it significantly. Qualitatively, the same results were obtained at 22°C and 35°C. Ouabain did not significantly affect the electrical activity of isolated, electrically stimulated myocytes, but it increased the amplitude of shortenings of these myocytes in a dose-dependent manner. Thus, the positive inotropic effect of ouabain at therapeutic doses (10 nM) occurs in spite of decreased Ca²⁺ content, while at high toxic doses the positive inotropic effect is accompanied by an increment in Ca²⁺ content. These data support the hypothesis that the mechanisms of positive inotropy of ouabain are different at therapeutic and toxic concentrations of this drug. Finally, our study demonstrates that the effects of low doses of ouabain are independent of the release of endogenous catecholamines.     

Pertussis-toxin-sensitive inhibition by (-) baclofen of Ca signals in bovine chromaffin cells

Ca signals in bovine adrenal chromaffin cells were studied both in Fura-2/AM-loaded intact cells, and in voltage-clamped cells under whole-cell patch-clamp conditions. The effects of gamma-aminobutyric acid b subtype (GABA_b) receptor activation on K⁺-depolarization-induced signals and on voltage-activated Ca²⁺ currents were investigated. Both GABA (20 M) plus bicuculline (20 M) and (-)baclofen (20–100 M), effectively inhibited the Ca signal in intact cells. The effects caused by baclofen continued to develop during the time interval between two successive stimuli. The restoration of the Ca signal during washout of baclofen was also delayed and continued in some experiments for 10–20 min. The inhibitory effect of baclofen on the Ca signal was eliminated by pre-treatment of the cells with pertussis toxin (PTX, 1g/ml, for 4–6h at 37°C). Baclofen (50 M) inhibited Ca²⁺ current in whole-cell mode by at most 20%. The effect developed quickly and was reversible. Infusion into the cells of a non-hydrolyzable analogue of guanosine 5-triphosphate GTP S (100 M), led to complete inhibition of the Ca²⁺ conductance and of voltage-evoked intracellular Ca ([CA]_i) transients within 2 min. In paired cells intracellularly perfused with GTPS-free solution, the Ca²⁺ current amplitude decreased by only about 30% for 5–6 min. It is concluded that bovine chromaffin cells have functional GABA_b receptors the activation of which, mediated by a PTX-sensitive GTP-binding protein, inhibits the evoked increase in cytosolic free Ca²⁺. The small size of the effect on Ca²⁺ current in whole-cell mode as compared to that on the Ca signal in intact cells may be explained by washout of some regulatory element during cell dialysis, or by a relatively small contribution of the normal voltage-activated Ca²⁺ current to the Ca signal. Alternatively, it might indicate GABA_b effects on mechanisms other than Ca²⁺ channels.     

Sugar transport in isolated rat kidney papillary collecting duct cells

d-Glucose is an important substrate of energy metabolism and osmolyte synthesis in the renal papillary collecting duct. In order to characterize the cellular entry ofd-glucose in this tubular segment, collecting duct cells were isolated from rat kidney papilla and the rate ofd-glucose uptake was measured indirectly by monitoring thed-glucose-dependent O₂ uptake in the presence of the uncoupler CCCP.d-Glucose uptake was found to be sodium-independent and not sensitive to phlorizin even at a concentration of 10^–3 M. Uptake was, however, completely inhibited by 10^–5 M cytochalasin B and 10^–4 M phloretin. The apparentK _i for cytochalasin B was 1.5×10^–6 M and for phloretin 2.0×10^–5 M. Studies on the substrate specificity revealed that at 1 mMd-mannose is taken up and metabolized to the same extent asd-glucose. A 50-fold higher concentration of 2-deoxy-d-glucose and 2-amino-2-deoxy-d-glucose inhibitedd-glucose uptake completely whereas -methyl-d-glucoside,d-allose, andd-galactose were without effect. Under conditions whered-glucose utilization was maximally stimulated an apparentK _m of 1.2 mM and aV _max of 1 mmold-glucose/g protein hour was found ford-glucose uptake.These results indicate that thed-glucose uptake into papillary collecting duct cells is probably mediated by a transport system similar to the one found in basal-lateral membranes of pelarized renal, intestinal, and liver cells as well as in nonpolarized fat cells and erythrocytes.Supported by grant DFG Gr 916/1-1     

Ca2+ not cyclic AMP mediates the fluid secretory response to isoproterenol in the rat mandibular salivary gland: Whole-cell patch-clamp studies

We have performed whole-cell patch-clamp studies on dispersed seccretory cells of the rat mandibular gland to determine how -adrenergic stimulation causes fluid secretion. When the pipette contained a high K⁺ solution, the resting membrane potential averaged –33 mV±1.1 (SEM,n=34) and the clamped cell showed strong outward rectification. We monitored K⁺ and Cl^– currents for periods of 15 min by recording the currents needed to clamp the cell potential at 0 and –80 mV, respectively. Isoproterenol (1–2 mol/l) caused increases in the clamp current at 0 mV (the K⁺ current) and at –80 mV (the Cl^– current) in about 80% of cases, although the responses were variable in size and time-course; the responses were indistinguishable from those induced by acetylcholine or the Ca²⁺ ionophore, A23187. The -adrenergic antagonist, phentolamine (1–2 mol/l), had no effect on the response, but the -adrenergic antagonist, propranolol (10 mol/l), blocked it completely. The isoproterenol response could not be mimicked by application to either surface of the cell membrane, of cyclic AMP (100 mol/l), forskolin (1 or 20 mol/l) or cholera toxin (2.5 g/ml). However, increasing the Ca²⁺-chelating capacity of the pipette solution by raising its EGTA concentration from the customary 0.5 to 20 mmol/l, blocked the response to isoproterenol, suggesting that -adrenergic agonists activate Cl^– and K⁺ channels by raising cytosolic Ca²⁺. Since neomycin, which blocks phospholipase C, blocked the action of isoproterenol without impairing the cell responsiveness to A23187, it appears that isoproterenol, like muscarinic agonists, increased cytosolic Ca²⁺ via the phosphatidylinositol cycle.This project was supported by the National Health and Medical Research Council of Australia     

Effects of T-type, L-type, N-type, P-type, and Q-type calcium channel blockers on stimulus-induced pre-and postsynaptic calcium fluxes in rat hippocampal slices

The contribution of T-, L-, N-, P-, and Q-type Ca²⁺ channels to pre-and postsynaptic Ca²⁺ entry during stimulus-induced high neuronal activity in area CA1 of rat hippocampal slices was investigated by measuring the effect of specific blockers on stimulus-induced decreases in extracellular Ca²⁺ concentration ([Ca²⁺]₀). [Ca²⁺]₀ was measured with ion-selective electrodes in stratum radiatum (SR) and stratum pyramidale (SP), while Ca²⁺ entry into neurons was induced with stimulus trains (20 Hz for 10 s) alternately delivered to SR and the alveus, respectively. The [Ca²⁺]₀ decreases recorded in SR in response to SR stimulation represented mainly presynaptic Ca²⁺ entry (Ca_pre), while [Ca²⁺]⁰ decreases recorded in SP in response to alvear stimulation were predominantly based on postsynaptic Ca²⁺ entry (Ca_post). Ethosuximide and trimethadione were ineffective m concentrations up to 1 mM. At 10 mM, they reduced Ca_post and, much less, also Ca_pre Nimodipine (25 M) reduced Ca_post and, to a minor extent, Ca_pre. -Agatoxin IVA (0.4–1 M) and -conotoxin MVIIC (1 M) also reduced both Ca_pre and Ca_post, but with a stronger action on Ca_pre. -Conotoxin GVIA (3–8 M) reduced Ca_post without effect on Ca_pre. We conclude that during stimulus-induced, high-frequency neuronal activity Ca_post is carried by P/Q-, N-, and L-type channels and probably a further channel type different from these channels. Ca_pre includes at least P/Q-and possibly L-type channels. N-type channels did not contribute to Ca_pre in our experiments. Since ethosuximide and trimethadione were only effective in high concentrations, their action may be unspecific. Thus, T-type channels do not seem to play a major part in Ca²⁺ entry in this situation.     

Sorbitol metabolism in inner medullary collecting duct cells of diabetic rats

Intracellular accumulation of sorbitol, generated fromd-glucose via the aldose reductase pathway, is thought to play an important role in diabetic complications such as lens cataracts and neuropathy. In order to elucidate the effect of diabetes on the renal inner medulla, another sorbitol-rich tissue, male Wistar rats were treated with a single dose of streptozotocin (60 mg/kg body weight, i.p.). Six wecks later total inner medullary tissue (IM) or isolated inner medullary collecting duct (IMCD) cells were prepared. In diabetic IM tissue, sorbitol content was 1.8-fold higher than in control IM tissue (134±17 vs. 74±22 mol/g tissue protein). Sorbitol production in both normal and diabetic IMCD cells was strongly dependent on extracellulard-glucose concentration. In normal cells, for example, sorbitol production was 90±9 mol sorbitol/g protein x h at 45 mMd-glucose compared to 13±1 mol/g protein x h at 5 mM. At identicald-glucose concentrations sorbitol synthesis in diabetic IMCD cells was, however, always significantly higher than in control cells (122% of control at 15 mM and 126% of control at 45 mM). In addition, aldose reductase activity in diabetic IM was found to be augmented. The maximal velocity was 4.2 times higher (97±22 U/g protein vs. 23±7 U/g protein) while theK _m of the enzyme remained unchanged. Membrane permeability for sorbitol or the response to changes in extracellular osmolarity was not significantly different in diabetic IMCD cells and normal cells with correspondingly high intracellular sorbitol concentrations. Similarly the kinetic parameters ofd-glucose uptake were not altered by streptozotocin treatment. These results suggest that increased medullary sorbitol content in diabetic rats is a result of increased sorbitol synthesis due to a higher extracellulard-glucose concentration and augmented aldose reductase activity in face of an unaltered sorbitol permeability of the plasma membrane.     

β-Adrenoceptor stimulation activates large-conductance Ca2+-activated K+ channels in smooth muscle cells from basilar artery of guinea pig

We studied the effect of isoproterenol on the Ca²⁺-activated K⁺(BK) channel in smooth muscle cells isolated from the basilar artery of the guinea pig. Cells were studied in a whole-cell configuration to allow the clamping of intracellular Ca²⁺ concentration, [Ca²⁺]_i. Macroscopic BK channel currents were recorded during depolarizing test pulses from a holding potential (V _H) of 0 mV, which was used to inactivate the outward rectifier. The outward macroscopic current available from aV _H of 0 mV was highly sensitive to block by external tetraethylammonium·Cl (TEA) and charybdotoxin, and was greatly augmented by increasing [Ca²⁺]_i from 0.01 to 1.0 M. With [Ca²⁺]_i between 0.1 and 1.0 M, 0.4 M isoproterenol increased this current by 58.6±17.1%, whereas with [Ca²⁺]_i at 0.01 M a sixfold smaller increase was observed. With [Ca²⁺]_i0.1 M, 100 M dibutyryl-adenosine 3:5: cyclic monophosphate (cAMP) and 1 M forskolin increased this current by 58.5±24.1% and 59.7±10.3%, respectively. The increase with isoproterenol was blocked by 4.0 M propranolol extracellularly, and by 10 U/ml protein kinase inhibitor intracellularly. Single-channel openings during depolarizing test pulses from aV _H of 0 mV recorded in the whole-cell configuration under the same conditions (outside-outwhole-cell recording) indicated a slope conductance of 260 pS. In conventional outside-out patches, this 260-pS channel was highly sensitive to block by external TEA, and in inside-out patches, its probability of opening was greatly augmented by increasing [Ca²⁺]_i from 0.01 to 1.0 M. Outside-out-whole-cell recordings with [Ca²⁺]_i0.1 M indicated that 100 M dibutyryl-cAMP increased the probability of opening of the 260-pS channel by 152±115%. In inside-out patches, the catalytic subunit of protein kinase A increased the probability of opening, and this effect also depended on [Ca²⁺]_i, with a 35-fold larger effect observed with 0.1–0.5 M Ca²⁺ compared to 0.01 M Ca²⁺. We conclude that the BK channel in cerebrovascular smooth muscle cells can be activated by-adrenoceptor stimulation, that the effect depends strongly on [Ca²⁺]_i, and that the effect is mediated by cAMP-dependent protein kinase A with no important contribution from a direct G-protein or phosphorylation-independent mechanism. Our data indicate that the BK channel may participate in-adrenoceptor-mediated relaxation of cerebral vessels, although the importance of this pathway in obtaining vasorelaxation remains to be determined.     

d-glucose balance in the enterocyte of rat jejunum “in vitro”

An everted sac of male albino rat jejunum (Wistar strain) incubated in vitro is used. Netd-glucose and Na⁺ transport together withd-glucose concentration in the emerging fluid [5], or in the serosal fluid, and in the enterocytes are determined. Celld-glucose concentration does not change significantly in a range between 20–200 moles or between 50–500 moles of netd-glucose transepithelial transport, depending on the experimental conditions.As far as cellulard-glucose and Na⁺ concentration is concerned, the enterocyte behaves as an homeostatic system.The mechanism involved ind-glucose extrusion is extensively discussed. Two hypotheses seem to be possible. First, the mechanism is an active metabolically dependent one, just as it is for sodium transport. Second, the metabolic activity favoursd-glucose facilitated permeability through the basolateral membrane in such a way as to maintain a constant relationship betweend-glucose and Na-extrusion, notwithstanding the fact thatd-glucose concentration gradient across the basolateral membrane lowers by increasing Na and glucose extrusion rate.     

Inhibition of L-type calcium channels by internal GTP [γS] in mouse pancreatic β cells

Pretreatment of pancreatic cells with pertussis toxin resulted in a 30% increase in peak whole-cell Ca²⁺ currents recorded in the absence of exogenous intracellular guanine nucleotides. Intracellular application of 90 M GTP[S], by liberation from a caged precursor, resulted in 40% reduction of the peak Ca²⁺ current irrespective of whether the current was carried by Ca²⁺ or Ba²⁺. Effects on the delayed outward K⁺ current were small and restricted to a transient Ca²⁺-dependent K⁺ current component. Inhibition by GTP[S] of the Ca²⁺ current was not mimicked by standard GTP and could not be prevented either by pretreatment with pertussis toxin or by inclusion of GDP[S] or cyclic AMP in the intracellular medium. The inhibitory effect of GTP[S] could be counteracted by a prepulse to a large depolarizing voltage. A similar effect of a depolarizing prepulse was observed in control cells with no exogenous guanine nucleotides. These observations indicate that inhibition of cell Ca²⁺ current by G protein activation results from direct interaction with the channel and does not involve second-messenger systems. Our findings also suggest that the cell Ca²⁺ current is subject to resting inhibition by G proteins.     

Control of pulmonary vascular smooth muscle tone by sarcoplasmic reticulum ca2+ pump blockers: thapsigargin and cyclopiazonic acid

The effect of thapsigargin (TG) and cyclopiazonic acid (CPA) on the mechanical activity of the rat pulmonary artery were investigated. In chemically (-escin)-skinned arterial strips, application of TG (0.1–1 M) or CPA (0.5–10 M) prior and throughout the loading procedure of the internal Ca²⁺ stores (0.3 M free Ca²⁺ ions for 8–10 min) concentration dependently inhibited the subsequent contractile response induced by noradrenaline (NA, 10 M) or caffeine (25 mM). In intact strips repeatedly incubated in a Ca²⁺-containing solution (2.5 mM for 10 min), followed by incubation in a Ca²⁺-free solution 12 min before NA-stimulation, TG and CPA not only inhibited the NA-induced contraction but also increased the tension which appeared during the exposure time to Ca²⁺. The two phenomena developed with similar time courses. The increase in tension during the readmission of Ca²⁺ ions was not antagonized by verapamil (10 M) or nifedipine (1 M) but was blocked by La³⁺ (50 M) and Co²⁺ (1 mM) ions. The amplitude of the verapamil-insensitive TG (or CPA)-induced contraction was dependent on the external [Ca²⁺] [0.1–10 mM, concentration for half maximal effect (EC₅₀) =0.85 mM], not modified by the reduction of the external [Na⁺] (from 130 to 10 mM) and decreased by depolarization of the strip using K⁺-rich (30–120 mM) solutions. Under the latter condition, 38±9 and 83±4% reduction (n=5) was observed in the presence of 60 and 120 mM K⁺ respectively. This contraction was also concentration dependently inhibited by the tyrosine kinase inhibitors genistein (0.5–50 M) and tyrphostin (2–50 M). Sr²⁺ ions, which contracted both depolarized intact and skinned strips, failed to replace Ca²⁺ ions in the verapamil-insensitive contraction induced by TG or CPA (n=4). Finally, TG (1 M) and CPA (10 M) did not modify the pCa tension relationship in skinned strips (n=5). These results show that the main action of TG and CPA in rat pulmonary artery is to prevent the refilling of the internal Ca²⁺ store. TG and CPA also seem to facilitate a Ca²⁺ influx through a specific verapamil-insensitive pathway. The biophysical and molecular characteristics of this pathway remain to be elucitated, although it appears to involve a tyrosine kinase activity.     

β-Adrenergic modulation of transient inward current in guinea-pig cardiac myocytes

Transient inward current (I_ti) indicating Ca²⁺-release from the sarcoplasmic reticulum and L-type Ca²⁺-current(I_Ca) were studied in atrial and ventricular myocytes from hearts of adult guinea-pigs by means of whole-cell voltage-clamp. The increase of I_Ca caused by -adrenergic stimulation using isoprenaline (ISO) or related experimental manoeuvres such as superfusion with forskoline (FORSK) was used as a qualitative monitor of an increase of intracellular cAMP. Changes of I_ti were used to manifest changes of sarcoplasmic Ca²⁺-release. In myocytes dialysed with citrate-based (60 mM) pipette filling solution containing 100 M EGTA spontaneous transient inward currents were recorded at a constant holding potential of –50 mV in the majority of myocytes. Superfusion with a solution containing ISO (5·10^–8M) increased the amplitude of spontaneous I_ti and reduced its time-to-peak. The effects of ISO on I_ti developed in parallel to stimulation of I_Ca. In myocytes which did not show spontaneous cyclic Ca²⁺-release in the above condition, this could be evoked de novo by ISO. Spontaneous I_ti was suppressed in the majority of cells by increasing the concentration of EGTA in the dialysing solution to 200 M. Brief (50 ms) activation of I_Ca by voltage steps from –50 to +10 mV usually failed to trigger Ca²⁺-release from the SR. The increase of I_Ca-amplitude upon administration of ISO went ahead with the induction of Ca²⁺-release by brief activation of I_Ca. The effects of ISO could be mimicked by FORSK or intracellular dialysis with 35-cyclic adenosine monophosphate. The effects on I_Ca and SR Ca²⁺-release were dependent on the concentration of the stimulating substance. In a given cell changing superfusion from a low to a high concentration of ISO or FORSK resulted in an increase of the number of Ca²⁺-release events per number of Ca²⁺-currents elicited and a shortening of time-to-peak of I_ti's. The stimulating effects of ISO or FORSK on Ca²⁺-release were only partially due to an increase of the triggering I_Ca. Ca²⁺-currents too small to trigger Ca²⁺-release before -adrenergic stimulation could evoke Ca²⁺-release after augmentation of intracellular cAMP. Whereas the effects of ISO and FORSK on I_Ca were reversible, the stimulatory effects on Ca²⁺-release persisted after washing out the substances. The results give support to the hypothesis that -adrenoceptor-mediated positive inotropic and arrhythmogenic effects are, at least partly, due to a cyclic AMP-dependent regulatory mechanism modulating sarcoplasmic Ca²⁺-release.This work was supported by the Deutsche Forschungsgemeinschaft (FG Konzell)