Role of actin microfilament in osmotic stretch-induced increase of voltage-operated calcium channel current in guinea-pig gastric myocytes

 Using the whole-cell patch clamp technique, the role of actin microfilament in hyposmotic increase of voltage-operated calcium channel current (I _Ba) was studied in guinea-pig gastric myocytes. Hyposmotic superfusate (212 mOsm) increased peak I _Ba amplitude by 32.7 ± 6.5%; when cytochalasin-D (Cyt-D, 20 μM), an actin cytoskeleton disruptor, was used, an increase of only 9.7 ± 3.1% was seen. I _Baresponse to osmotic stress was potentiated (45.1 ± 4.1% increase) by 20 μM phalloidin, an actin microfilament stabilizer. However, colchicine (100 μM), an microtubule cytoskeleton disruptor, had no effect on either I _Ba or its response to hyposmotic solution. Phalloidin also induced a rightward shift of the I/V relationship of I _Ba, while Cyt-D itself had no effect. These results suggest that actin cytoskeleton may mediate hyposmotic stretch-induced I _Ba increase in gastric smooth muscle. Received: 26 March 1997 / Received after revision: 28 May 1997 / Accepted: 3 June 1997     

Membrane responses to extracellular ATP in rat isolated white adipocytes

 We used whole-cell and perforated-patch voltage-clamp methods to study the membrane electrical properties of isolated rat epididymal and inguinal white adipocytes. We examined cells from both Sprague-Dawley and Zucker lean and Zucker obese (fa/fa) rats. A delayed-rectifier potassium current was present and similar in unstimulated white fat cells from all these sources. The potassium current activated rapidly with depolarization positive to about –30 mV and showed slow inactivation. Stimulation with extracellular ATP activated both hyperpolarizing and depolarizing conductances. ATP exposure also increased cell membrane capacitance by an average of 16%, suggesting that ATP activates exocytosis. Exposure to norepinephrine had little electrophysiological effect. We conclude that white adipocytes are very similar to brown adipocytes in their resting electrophysiological profile and in their responses to extracellular ATP. Received: 2 January 1997 / Received after revision: 14 April 1997 / Accepted: 15 April 1997     

The sarcolemmal mechanisms involved in the control of diastolic intracellular calcium in isolated rat cardiac trabeculae

 We performed experiments using the calcium indicator Indo-1 to determine the relative roles of the sarcolemmal mechanisms involved in the regulation of diastolic intracellular calcium concentration ([Ca²⁺]_i) in trabeculae from the rat heart. Ryanodine was used to eliminate sarcoplasmic reticulum (SR) function. In the functional absence of the SR, 76.8 ± 3.9% of the calcium was extruded by the Na-Ca exchange carrier in the [Ca²⁺]_i range of diastolic concentration ± 200–400 nM. This was assessed by measuring the recovery of [Ca²⁺]_i from small perturbations in the presence and absence of extracellular sodium. The steady-state relationship between [Ca²⁺]_o and [Ca²⁺]_i was linear over the range of 1–40 mM, a 20-fold increase of [Ca²⁺]_o produced a 1.97-fold ± 0.13-fold increase in [Ca²⁺]_i (n = 5). In the absence of extracellular sodium raising [Ca²⁺]_o had a variable effect. In some preparations there was little change of [Ca²⁺]_i while in others the response was almost as large as in control conditions. We conclude that the Na-Ca exchanger contributes ≈ 77% of sarcolemmal calcium extrusion following small perturbations in [Ca²⁺]_i and that this fraction does not diminish as the [Ca²⁺]_i declines. In addition we have shown a sodium-independent entry of calcium into quiescent cardiac muscle under resting conditions. Received: 16 May 1996 / Received after revision and accepted: 28 June 1996     

The effects of inhibition of the sarcolemmal Ca-ATPase on systolic calcium fluxes and intracellular calcium concentration in rat ventricular myocytes

 The effects of carboxyeosin, an inhibitor of the sarcolemmal Ca-ATPase, were studied on intracellular Ca and membrane currents in isolated rat ventricular myocytes. In the absence of carboxyeosin, 150-ms-duration depolarizing pulses from –40 to 0 mV resulted in an L-type Ca current on depolarization and a Na-Ca exchange ”tail” current on repolarization. The calculated entry of Ca on the L-type current was 1.3 times greater than the efflux via the Na-Ca exchange. The addition of carboxyeosin (20 μM) resulted in either an increase of the Na-Ca exchange current or a decrease of the L-type Ca current such that the Ca entry and efflux were exactly equal. These results suggest that, under control conditions, a carboxyeosin-sensitive Ca-ATPase contributes about 24% of the total Ca efflux from the cell and, therefore, that the sarcolemmal Ca-ATPase has a significant role in regulation of sarcolemmal Ca fluxes. Received: 9 December 1998 / Received after revision: 1 February 1999 / Accepted: 2 February 1999     

Comparison of the postprandial release of peptide YY and proglucagon-derived peptides in the rat

Endocrine L-cells of the distal intestine synthesize both peptide YY (PYY) and proglucagon-derived peptides (PGDPs), whose release has been reported to be either parallel or selective. Here we compare the release mechanisms of PYY, glucagon-like peptide-1 (GLP-1), and oxyntomodulin-like immunoreactivity (OLI) in vivo. Anaesthetized rats were intraduodenally (ID) given either a mixed semi-liquid meal or oleic acid, or they received oleic acid or short chain fatty acids (SCFA) intracolonically (IC). The ID meal released the three peptides with a similar time-course (peak at 30 min); ID oleic acid produced a progressive release of PYY and OLI, while GLP-1 release was less. IC oleic acid or SCFA released smaller (but significant) amounts of PYY but no OLI or GLP-1. Hexamethonium inhibited most of the response to the ID meal and ID oleic acid, but did not change the PYY response to IC oleic acid. N ^G -nitro-l-arginine methyl ester (l-NAME, a nitric oxide synthase inhibitor) inhibited meal-induced PYY release and left OLI and GLP-1 unaffected. BW10 (a gastrin-releasing peptide antagonist) had no effect on the meal-induced release of either peptide. These results suggest a parallel initial release of PYY, OLI and GLP-1 after the ID meal, or oleic acid, by an indirect mechanism triggered in the proximal bowel, using nicotinic synapses, and involving nitric oxide release for PYY and an unknown mediator for PGDPs. For PYY there is a later phase of peptide release, probably induced by direct contact between nutrients and colonic L-cells. Received: 8 January 1999 / Received after revision: 25 March 1999 / Accepted: 26 April 1999     

Effect of ryanodine on atrial natriuretic peptide secretion by contracting and quiescent rat atrium

To elucidate the mechanism involved in the release of atrial natriuretic peptide (ANP), we studied the importance of ryanodine-sensitive Ca²⁺ release in stretch-secretion coupling. The experiments were made with a left atrial preparation, where the stretch of myocytes was induced by changing the intra-atrial pressure. When external pacing was not applied, the atrial preparation was not spontaneously contracting, and it was therefore possible to investigate the secretory mechanism in the quiescent atrium. The superfusate was collected in 2-min fractions and assayed for ANP immunoreactivity. Filtration analysis revealed that the major fraction in the superfusate in all experimental situations had a similar molecular weight as the ANP 1–28. Ryanodine (1.0 M and 0.1 M) inhibited stretch-stimulated ANP secretion dose dependently both in paced and nonpaced atrium, but did not have any effect on basal secretion. The present results support the notion that intracellular Ca²⁺ transients from the intracellular stores are essential for stretch-stimulated ANP secretion, independently from excitation and contraction. Basal ANP secretion is not inhibited by blocking ryanodine-sensitive Ca²⁺ channels, either in contracting or in non-contracting atria. In addition our results confirm that the principal stimulus for ANP secretion in response to atrial distension is the stretch of myocytes. Length shortening of myocytes is not essential for ANP release.     

Rapid effects of cytochalasin-D on contraction and intracellular calcium in single rat ventricular myocytes

 Contraction and intracellular calcium ([Ca²⁺]_i) transients were recorded using a video edge detector and fluorescence spectrophotometry, respectively, in rat ventricular myocytes at 22–24°C stimulated at a frequency of 1 Hz. Application of the F-actin disrupter cytochalasin-D (Cyt-D) caused a large reduction in the amplitude of contraction and a small increase in the [Ca²⁺]_i transient. These responses began within a few seconds of application and were complete after 2 min of exposure. Phase-plane relationships of contraction and [Ca²⁺]_i were consistent with cytochalasin-D causing a decrease in myofilament responsiveness to Ca²⁺. Received: 18 May 1998 / Received after revision: 8 July 1998 / Accepted: 13 July 1998     

Angiotensin-II stimulates nitric oxide release in isolated perfused renal resistance arteries

 Nitric oxide (NO) has been implicated as a modulator of the vascular effects of angiotensin II (ANG II) in the kidney. We used a NO-sensitive microelectrode to study the effect of ANG II on NO release, and to determine the effect of selective inhibition of the ANG II subtype I receptor (AT1) with losartan (LOS) and candesartan (CAN). NO release from isolated and perfused renal resistance arteries was measured with a porphyrin-electroplated, carbon fiber. The vessels were microdissected from isolated perfused rat kidneys and perfused at constant flow and pressure in vitro. The NO-electrode was placed inside the glass collection cannula to measure vessel effluent NO concentration. ANG II stimulated NO release in a dose-dependent fashion: 0.1 nM, 10 nM and 1000 nM ANG II increased NO-oxidation current by 85±18 pA (n = 11), 148±22 pA (n = 11), and 193±29 pA (n = 11), respectively. These currents correspond to changes in effluent NO concentration of 3.4±0.5 nM, 6.1±1.1 nM, and 8.2±1.3 nM, respectively. Neither LOS (1 μM) nor CAN (1 nM) significantly affected basal NO production, but both AT1-receptor blockers markedly blunted NO release in response to ANG II (10 nM): 77±6% inhibition with LOS (n = 8) and 63±9% with CAN (n = 8). These results are the first to demonstrate that ANG II stimulates NO release in isolated renal resistance arteries, and that ANG II-induced NO release is blunted by simultaneous AT1-receptor blockade. Our findings suggest that endothelium-dependent modulation of ANG II-induced vasoconstriction in renal resistance arteries is mediated, at least in part, by AT1-receptor-dependent NO release. Received: 24 September 1997 / Accepted: 20 October 1997     

Role of physiological HCO3 –buffer on intracellular pH and histamine release in rat peritoneal mast cells

 The purpose of this study was to examine how intracellular pH (pH_i) regulation and histamine release are affected by HCO₃ ^–in rat peritoneal mast cells. The pH_i was measured using the pH-sensitive dye 2′, 7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). We observed a pH_i of 6.88±0.012 (n=24) in resting mast cells exposed to a HEPES buffer (pH 7.4), but a sustained drop of 0.21 pH units to 6.67±0.015 (n=23) when we exposed the mast cells to a HEPES/HCO₃ ^–buffer equilibrated at all time with 5% CO₂ (pH 7.4). This fall in pH_i is inhibited by the carbonic anhydrase inhibitor dichlorphenamide and is Na⁺-independent, indicating the involvement of Na⁺-independent Cl^–/HCO₃ ^–exchange activity. Furthermore removal of external Cl^–in the presence but not in the absence of HCO₃ ^–reversed the Cl^–/HCO₃ ^–exchange and induced an alkaline load. The recovery from this alkaline load was dependent on external Cl^–but independent of Na⁺. Both the alkalinization and the recovery were inhibited by the anion transport inhibitor 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS). In addition, ³⁶Cl^–uptake measurements confirm the presence of a Cl^–/HCO₃ ^–exchanger. Histamine release stimulated by antigen and compound 48/80 was substantially reduced in the presence of HEPES/ HCO₃ ^–buffer (pH_o 7.4, pH_i 6.66). Histamine release was increased, however, when pH_i was clamped to 6.66 in HCO₃ ^–-free media (pH_o 6.9). We conclude that: (1) Na⁺-independent Cl^–/HCO₃ ^–exchange determines steady-state pH_i in rat peritoneal mast cells; and (2) the reduction in histamine release observed in the presence of HCO₃ ^–is not due to its effect on pH_i per se, but rather on other changes in ion transport. Received: 29 January 1998 / Received after revision and accepted: 3 April 1998     

Intracellular pH and calcium signalling in rat pancreatic acinar cells

 Intracellular free Ca²⁺ signals, which occur in many secretory cell types after the binding of some secretagogues to their membrane receptors, are due to Ca²⁺ mobilization from internal stores and Ca²⁺ influx from the extracellular space. There is also growing evidence for a modulatory role of intracellular pH in Ca²⁺ metabolism. In fact it has been proposed that Ca²⁺ stores in pancreatic acinar cells may be loaded by Ca²⁺/H⁺ exchange. The aim of this paper was to establish the effect of intracellular pH on Ca²⁺ signalling in pancreatic acinar cells. Application of the proton carrier nigericin impairs Ca²⁺ mobilization in response to cholecystokinin (CCK-8), and application of membrane-permeant bases or acids inhibits CCK-8-evoked intracellular Ca²⁺ oscillations. Both nigericin and a cell-permeant weak base release Ca²⁺ from internal stores. However, cytosolic acidification by removal of extracellular Na⁺ had no effect on the resting or stimulated cytosolic Ca²⁺ concentration. After depletion of Ca²⁺ stores by a maximal concentration of CCK-8, nigericin and ionomycin released a residual Ca²⁺ pool. Taken together, our results show that in pancreatic acinar cells Ca²⁺ signals require the existence of subcellular gradients of pH and indicate the presence of acidic pools of Ca²⁺. Received: 21 March 1997 / Received after revision: 14 May 1997 / Accepted: 15 May 1997     

Capacitative calcium entry is colocalised with calcium release in Xenopus oocytes: Evidence against a highly diffusible calcium influx factor

 Depletion of intracellular calcium stores activates the plasma membrane capacitative calcium entry pathway in many cell types. The nature of the signal that couples the depletion of the intracellular calcium stores to the activation of the plasma membrane calcium influx pathway is as yet unknown. It has recently been suggested that a highly diffusible calcium influx factor is involved in the activation of capacitative calcium entry, and that its action is potentiated by the protein phosphatase inhibitor okadaic acid. Depletion of intracellular calcium stores in a localised region of a Xenopus oocyte was found to evoke capacitative calcium entry exclusively colocalised across the stimulated area of the plasma membrane, arguing against the involvement of a highly diffusible calcium influx factor. Equally, no evidence could be found for the presence of a soluble calcium influx factor in the bulk cytosol of Xenopus oocytes. The potentiation of capacitative calcium entry by okadaic acid resembled that mediated by the activation of protein kinase C, thus suggesting that okadaic acid activity may not necessarily be related to the action of a putative calcium influx factor. Received: 2 January 1996 / Received after revision and accepted: 14 February 1996     

Ca2+-channel-dependent and -independent inhibition of exocytosis by extracellular ATP in voltage-clamped rat adrenal chromaffin cells

 Membrane currents and capacitance were measured to examine the effects of extracellular ATP on exocytosis in voltage-clamped rat adrenal chromaffin cells. ATP reversibly inhibited Ca²⁺ current (I _Ca) and exocytosis. The dependency of exocytosis on I _Ca evoked by 1-s depolarizations was determined. However, inhibition of exocytosis was 2.6 times larger than that estimated from the reduction of I _Ca, implying the existence of a Ca²⁺-channel-independent pathway. This inhibition did not rely on a further reduction of the intracellular Ca²⁺ concentration spike. ATP reduced the rate of exocytosis induced by clamping the intracellular Ca²⁺ concentration. Pertussis toxin blocked the inhibitory effects of ATP on I _Ca and exocytosis. Although RB-2, a P_2Y antagonist, blocked the inhibitory effect of ATP on I _Ca, RB-2 itself produced large increase or decrease in membrane capacitance. Adenosine inhibited I _Ca via a pertussis-toxin-sensitive pathway but did not significantly inhibit exocytosis. Our data show that extracellular ATP inhibits exocytosis via inhibition of I _Ca by activation of a pertussis-toxin-sensitive G-protein linked to P_2Y receptors. Furthermore, our data strongly suggest that ATP activates another pathway, which is also G-protein dependent and accounts for the majority of the inhibitory effect of ATP on exocytosis. Received: 20 February 1997 / Received after revision: 10 July 1997 / Accepted: 23 July 1997     

2,3-Butanedione monoxime (BDM) decreases sarcoplasmic reticulum Ca content by stimulating Ca release in isolated rat ventricular myocytes

 The effects of 2,3-butanedione monoxime (BDM) were examined using rat ventricular myocytes loaded with Indo-1 to measure the intracellular Ca concentration ([Ca²⁺]_i). BDM (10 mM) produced a transient increase of the systolic Ca transient with no steady-state effect on its magnitude. This transient increase was more marked when BDM was applied after having decreased the external Ca concentration from 1 to 0.1 mM. There was a transient increase of resting [Ca²⁺]_i in both quiescent and electrically stimulated cells. Prior application of BDM decreased the rise of [Ca²⁺]_i produced by caffeine. In voltage-clamped cells the rise of [Ca²⁺]_i produced by BDM was accompanied by a transient inward current attributed to the electrogenic Na-Ca exchange. The amount of Ca lost from the cell upon application of 10 mM BDM could be estimated either from the integral of the BDM-evoked current or from the reduction of the integral of a caffeine-evoked current and corresponded to about 50% of the sarcoplasmic reticulum (s.r.) Ca content. The decrease of s.r. Ca content and the transient potentiation of the systolic Ca transient suggest that BDM acts by stimulating Ca-induced Ca release. These effects must be allowed for when using BDM. Received: 27 March 1998 / Received after revision: 12 May 1998 / Accepted: 13 May 1998     

Myogenin mRNA is elevated during rapid, slow, and maintenance phases of stretch-induced hypertrophy in chicken slow-tonic muscle

 Biochemical changes that are associated with the growth phase of stretch-induced skeletal muscle hypertrophy are better understood than events that maintain the increased muscle mass. One purpose of this study was to determine whether changes that occur during the period of rapid muscle hypertrophy persist during periods when muscle growth plateaus or the rate of enlargement slows. Serum response factor (SRF), myogenin, MyoD, and actin mRNA expression patterns were examined. SRF protein interactions with serum response element-1 (SRE1) of the chicken skeletal α-actin gene were also characterized. Anterior latissimus dorsi (ALD) wet weight (132% and 122%) and total RNA concentration (29% and 19%) increased after 2 and 3 weeks of stretch overload, respectively. Myogenin mRNA per microgram RNA increased after 3 (775%), 6 (1073%), 14 (227%), and 21 days (133%) of stretch overload. At 6 days, myogenin mRNA levels were increased in the distal, middle and proximal regions of the ALD. Serum response factor (SRF) mRNA per microgram total RNA was not increased after 2 or 3 weeks of stretch overload. MyoD and skeletal α-actin mRNAs per microgram total RNA were also unchanged after 2 and 3 weeks of stretch. Gel mobility shift assays demonstrated that SRF bound to SRE1 from 14-day-stretched ALD nuclear extracts had an increased mobility compared to control, and this difference in mobility was maintained in nuclear extracts from ALD muscle whose mass was declining. These results indicate that the expression of myogenin mRNA and total RNA remains elevated during either slow or maintenance periods of stretch-induced increases in ALD mass, when SRF mRNA has returned to control levels. Additionally, stretch-induced alterations in SRF binding to SRE1, from the skeletal α-actin promoter, occur regardless of the rate of stretch-induced growth. Received: 26 June 1997 / Received after revision: 25 November 1997 / Accepted: 26 November 1997     

Regulation of ionic currents by protein kinase A and intracellular calcium in outer hair cells isolated from the guinea-pig cochlea

 Two prominent potassium currents, termed I _K and I _K,n, and a cation current are found in outer hair cells (OHCs) of the guinea-pig cochlea. We report here whole-cell recordings which indicate that the currents are regulated by intracellular factors. 8-bromo-cAMP (500 μM), a membrane-permeable cAMP analogue, activated potassium currents in OHCs in both apical and basal turns of the cochlea. In OHCs from the cochlear apex, the drug effect was largest at potentials positive to –40 mV, indicating I _K as the target. In short cells from the cochlear base, both I _K and I _K,n were affected. The effects of 8-bromo-cAMP could be blocked by the presence of 1 μM H-89 (a protein kinase A inhibitor) in the patch pipette solution. Extracellular application of 10 nM okadaic acid, a protein phosphatase inhibitor, also activated both potassium currents. Currents were also modulated by intracellular calcium. I _K was activated in long cells by photorelease of calcium from the caged compound nitr5. Cation current activation required calcium release by photolysis of DM-nitrophen, a compound releasing more calcium. The results show that OHC potassium channels are regulated by background phosphorylation through protein kinase A and dephosphorylation by protein phosphatase. Cellular calcium also activates I _K and the cation channel, but with different sensitivities. Received: 1 September 1998 / Received after revision: 21 October 1998 / Accepted: 22 October 1998     

Endothelin-induced vasoconstriction and release of atrial natriuretic peptides in the rat

Endothelin-1 (ET-1) was given to male Sprague-Dawley rats in i.v. bolus injections to evaluate its effects on blood pressure and the release of atrial natriuretic peptides (ANP). In awake rats ET-1 (0.3, 1 and 3 nmol kg-1 body wt) transiently reduced mean arterial pressure (MAP) and increased heart rate (HR), followed by a prolonged increase in MAP. The magnitude of these changes and the duration of the increase in MAP were dose-related. The increase in MAP was completely blocked by verapamil, reversed by sodium nitroprusside, slightly reduced by rat atrial natriuretic factor (103-126) and unaffected by saralasin. The initial fall in MAP was also unaltered by these agents. In all groups HR changes were mirror-images of MAP. In anaesthetized rats ET-1 (1 nmol kg-1 body wt) induced a sustained release of ANP. Right atrial pressure increased transiently and then fell below baseline. When the increase in MAP was blocked with sodium nitroprusside, ET-1 still produced an increase in ANP. In conclusion we find that repeated i.v. administration of ET-1 induces immediate vasodilatation, without signs of tachyphylaxis, followed by long-lasting severe vasoconstriction. Baroreceptor function seems to be unchanged. ET-1 appears to induce ANP release by a direct action on atrial myocytes, independent of right atrial and systemic arterial pressure. We hypothesize that endothelin may be a mediator of stretch-induced release of ANP.     

Human adrenal chromaffin cell calcium channels: drastic current facilitation in cell clusters, but not in isolated cells

 Human adrenal medullary chromaffin cells were prepared and cultured from a cystic tumoral adrenal gland whose medullary tissue was unaffected. Adrenaline-containing and noradrenaline-containing cells were identified using a confocal fluorescence microscope and antibodies against dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT). Current/voltage (I/V) curves performed with the voltage-clamped cells bathed in 10 mM Ba²⁺ (holding potential, V _h=–80 mV) revealed the presence of only high-threshold voltage-dependent Ca²⁺ channels; T-type Ca²⁺ channels were not seen. By using supramaximal concentrations of selective Ca²⁺ channel blockers, the whole-cell I _Ba could be fractionated into various subcomponents. Thus, I _Ba had a 25% fraction sensitive to 1 μM nifedipine (L-type channels), 21% sensitive to 1 μM ω-conotoxin GVIA (N-type channels), and 60% sensitive to 2 μM ω-agatoxin IVA (P/Q-type channels). The activation of I _Ba was considerably slowed down, and the peak current was inhibited upon superfusion with 10 μM ATP. The slow activation and peak current blockade were reversed by strong depolarizing pre-pulses to +100 mV (facilitation). A drastic facilitation of I _Ba was also observed in voltage-clamped human chromaffin cell surrounded by other unclamped cells; in contrast, in voltage-clamped cells not immersed in a cell cluster, facilitation was scarce. So, facilitation of Ca²⁺ channels in a voltage-clamped cell seems to depend upon the exocytotic activity of neighbouring unclamped cells, which is markedly increased by Ba²⁺. It is concluded that human adrenal chromaffin cells mostly express P/Q-types of voltage-dependent Ca²⁺ channels (60%). L-Type channels and N-type channels are also expressed, but to a considerably minor extent (around 20% each). This dominance of P/Q-type channels in human chromaffin cells clearly contrasts with the relative proportion of each channel type expressed by chromaffin cells of five other animal species studied previously, where the P/Q-type channels accounted for 5–50%. The results also provide strong support for the hypothesis that Ca²⁺ channels of human chromaffin cells are regulated in an autocrine/paracrine fashion by materials co-secreted with the catecholamines, i.e. ATP and opiates. Received: 1 May 1998 / Received after revision and accepted: 21 May 1998     

ANP gene expression in rat hearts during hypoxia

 It is unclear whether the increase in plasma atrial natriuretic peptide (ANP) concentration during hypoxia is due to direct, hypoxia-induced upregulation of ANP secretion in the heart, or to pressure overload of the right ventricle (RV) following hypoxia-induced pulmonary hypertension. To test the hypothesis that hypoxia leads to an early upregulation of the ANP gene, we examined the influence of acute and prolonged inspiratory hypoxia (6 h, 1 or 3 weeks) on the expression of ANP messenger ribonucleic acid (mRNA) in rat heart and compared the results with the expression of the ANP gene after acute pressure overload induced by experimental coarctation of the main pulmonary artery. As a molecular marker for hypertrophy we determined the ratio of α- and β-myosin gene expression. Hypoxia increased systolic RV pressure from 20.0 ± 1.6 mmHg to 27.8 ± 1.6 mmHg (P < 0.01) and 41.6 ± 2.1 mmHg (P < 0.05) after 1 and 3 weeks hypoxia respectively. The ANP plasma concentration did not change significantly after 6 h or 1 week: 232 ± 21 pg/ml (control), 246 ± 25 pg/ml (6 h), 268 ± 25 pg/ml (1 week), but increased significantly after 3 weeks hypoxia (446.8 ± 99.56 pg/ml; P < 0.05). ANP mRNA levels in different regions of the heart did not change after 6 h or 1 week hypoxia. After 3 weeks hypoxia ANP mRNA had increased 2.7-fold in the RV (P < 0.05), 4.2-fold in the left ventricle (LV, P < 0.05), 3.5-fold in the septum (S, P < 0.05) and about 1.4-fold in the right (n.s.) and left atrium (n.s.). Relative ventricular masses increased significantly only for the RV (190%, P < 0.05) during hypoxia. The β/α-myosin mRNA ratio did not change after 6 h hypoxia but, contrary to ANP gene expression, increased after just 1 week (6.1-fold in RV, 7.8-fold in LV, 6-fold in S; P < 0.05) and was more pronounced in the RV after 3 weeks (9.4-fold in RV, 7.6-fold in LV, 9.1-fold in S; P < 0.05). The increase in the β/α-myosin mRNA ratio in the LV contrasts with a lack of increase in relative ventricular mass. Acute pressure overload in the RV after pulmonary arterial banding significantly increased ANP-mRNA and the β/α-myosin mRNA ratio after 1 day in the RV. In the LV ANP mRNA was unchanged. The delayed upregulation of the ANP gene suggests that hypoxia per se is not a significant stimulus for ANP gene expression in the heart and that hypoxia-induced ANP-gene expression in the heart is regulated predominantly by the increase in RV afterload due to hypoxia-induced increased pulmonary pressure. The upregulation of ANP and β-myosin mRNA in the LV during chronic hypoxia has yet to be elucidated. Received: 5 November 1996 / Received after revision and accepted: 24 January 1997     

Functional expression of rat brain cloned α1E calcium channels in COS-7 cells

 The properties of the rat brain α1E Ca²⁺ channel subunit and its modulation by accessory rat brain α2-δ and β1b subunits were studied by transient transfection in a mammalian cell line in order to attempt to reconcile the debate as to whether α1E forms a low-voltage-activated (LVA) or high-voltage-activated (HVA) Ca²⁺ channel and to examine its pharmacology in detail. α1E alone was capable of forming an ion-conducting pore in COS-7 cells. The properties of heteromultimeric α1E/α2-δ/β1b channels were largely dictated by the presence of the β1b subunit, which increased current density and tended to produce a hyperpolarizing shift in the voltage dependence of activation and inactivation. α1E/α2-δ/β1b channels did not appear to be regulated by Ca²⁺-induced inactivation. α1E was shown to exhibit a unique pharmacological profile. ω-Agatoxin IVA blocked the current in a dose-dependent manner with an IC₅₀ of approximately 50 nM and a maximum inhibition of about 80%, whilst ω-conotoxin MVIIC was without effect. The 1,4-dihydropyridine (DHP) antagonist nicardipine (1 μM) produced an inhibition of 51 ± 7%, whereas the DHP agonist S-(–)BAY K 8644 was without effect. Our findings suggest a re-evaluation of the classification of the α1E Ca²⁺ channel subunit; we propose that rat brain α1E forms a novel Ca²⁺ channel with properties more similar to a subtype of LVA than HVA Ca²⁺ current. Received: 30 August 1996 / Received after revision and accepted: 28 October 1996