The effect of propofol on angiotensin II-induced Ca(2+) mobilization in aortic smooth muscle cells from normotensive and hypertensive rats

We studied the effect of propofol (5.6-560 micromol/L; 1-100 microg/mL) on the mechanisms involved in Ca(2+) mobilization elicited by angiotensin II (AngII) in Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats. We studied the variations in intracellular Ca(2+) ([Ca(2+)](i)) concentrations in cultured aortic vascular smooth muscle cells (VSMCs) isolated from 6-wk-old WKY and SHR rats loaded with the Ca(2+)-sensitive fluorescent dye, Fura-2, using fluorescent imaging microscopy. In the absence of external Ca(2+), AngII (1 micromol/L) induced a transient [Ca(2+)](i) mobilization from internal stores that was larger in SHR than in WKY rats. Ca(2+) influx was assessed after external Ca(2+) (1 mmol/L) reintroduction. Propofol (1-100 microg/mL) added 5 min before the experiments did not alter AngII-induced Ca(2+) release from internal stores in either strain. By contrast, Ca(2+) influx elicited by AngII was significantly decreased by propofol. This effect occurred at a smaller concentration of propofol in the SHR than in the WKY rats. When Ca(2+) stores were depleted by exposure of cells to thapsigargin, an inhibitor of the sarcoendoplasmic reticulum Ca(2+)-ATPase, reintroduction of Ca(2+) to the medium induced a capacitative Ca(2+) influx of similar magnitude than that elicited by AngII. This influx was also significantly decreased by propofol at 100 microg/mL ( WKY: 27 +/- 3% of control values, n = 107; SHR: 16 +/- 3%, n = 47; P < 0.001). In conclusion, propofol decreased AngII-induced Ca(2+) influx through voltage-independent channels, without altering Ca(2+) release from internal stores in aortic VSMCs. The hypertensive rats were found to be more sensitive to the effect of propofol than the normotensive rats. This suggests that the response of VSMCs to AngII may be altered by propofol. IMPLICATIONS: In rat aortic vascular smooth muscle cells, propofol reduced angiotensin II-elicited Ca(2+) entry through capacitative Ca(2+) channels without altering Ca(2+) release from intracellular stores. Spontaneously hypertensive rats were more sensitive to these effects of propofol than normotensive rats. The response of vascular smooth muscle cells to angiotensin II may be altered by propofol.     

瑞芬太尼对正常和高血压大鼠基底动脉平滑肌细胞的不同作用

目的评价瑞芬太尼对正常和高血压大鼠基底动脉平滑肌细胞上大电导钙激活钾通道(BKCa)和电压门控钾通道(Kv)激活电流的影响。方法自发性高血压大鼠(spontaneously hypertensive rats,SHR)和同源正常血压(wistar-kyoto,WKY)大鼠,采用酶消化法急性分离基底动脉平滑肌细胞,每种大鼠选择6个基底动脉平滑肌细胞,采用全细胞膜片钳技术记录外向电流幅度。加入瑞芬太尼3×10-7mol/L,分别记录所设置的方波刺激(step刺激)方案中所有刺激电压下给药前(基础水平)和给药后电流幅度,并计算净电流=给药后电流幅度-基础值;采用浓度累积法给药,分别记录+60 m V刺激电压下给药前(基础值)和给予10-10、10-9、10-8、10-7、10-6、10-5mol/L瑞芬太尼后电流幅度,计算电流增加率和瑞芬太尼增加基底动脉平滑肌细胞电流幅度的半数有效浓度(EC50);另取每种大鼠6个基底动脉平滑肌细胞,加入瑞芬太尼3×10-7mol/L后分别给予BKCa阻滞剂四乙胺(tetraethylammonium,TEA)和Kv阻滞剂4-氨基吡啶(4-aminopyridine,4-AP),再分别加入其相应的瑞芬太尼混合液,记录每一次给药后的电流幅度。结果两种大鼠基底动脉平滑肌细胞给瑞芬太尼后在0、+20、+40和+60 m V刺激电压下产生的净电流依次明显增大(P0.05);10-10、10-9、10-8、10-7mol/L瑞芬太尼作用下,两种大鼠基底动脉平滑肌细胞外向电流增加率依次明显升高(P0.05);与WKY大鼠比较,瑞芬太尼增加SHR基底动脉平滑肌细胞电流幅度的(EC50)明显升高(P0.05);与基础值比较,两种大鼠基底动脉平滑肌细胞瑞芬太尼给药后电流幅度明显升高,TEA给药后或4-AP给药后电流幅度明显降低(P0.05);与TEA给药后或4-AP给药后比较,TEA+瑞芬太尼给药后或4-AP+瑞芬太尼给药后两种大鼠基底动脉平滑肌细胞电流幅度明显升高(P0.05)。结论瑞芬太尼呈电压依赖性和浓度依赖性激活两种大鼠基底动脉平滑肌细胞BKCa和Kv电流,瑞芬太尼对SHR基底动脉平滑肌细胞上BKCa和Kv激活电流的作用较WKY大鼠弱。     

Effects of volatile anesthetics on store-operated Ca(2+) influx in airway smooth muscle

BACKGROUND: In airway smooth muscle (ASM), volatile anesthetics deplete sarcoplasmic reticulum (SR) Ca(2+) stores by increasing Ca(2+) "leak." Accordingly, SR replenishment becomes dependent on Ca(2+) influx. Depletion of SR Ca(2+) stores triggers Ca(2+) influx via specific plasma membrane channels, store-operated Ca(2+) channels (SOCC). We hypothesized that anesthetics inhibit SOCC triggered by increased SR Ca(2+) "leak," preventing SR replenishment and enhancing ASM relaxation. METHODS: In porcine ASM cells, SR Ca was depleted by cyclopiazonic acid or caffeine in 0 extracellular Ca(2+), nifedipine and KCl (preventing Ca(2+) influx through L-type and SOCC channels). Extracellular Ca(2+) was rapidly introduced to selectively activate SOCC. After SOCC activation, SR was replenished and the protocol repeated in the presence of 1 or 2 minimum alveolar concentration halothane, isoflurane, or sevoflurane. In other cells, characteristics of SOCC and interactions between acetylcholine (Ach) and volatile anesthetics were examined. RESULTS: Cyclopiazonic acid produced slow SR leak, whereas the caffeine response was transient in ASM cells. Reintroduction of extracellular Ca(2+) rapidly increased [Ca(2+)]i. This influx was insensitive to nifedipine, SKF-96365, and KBR-7943, inhibited by Ni and blockade of inositol 1,4,5-triphosphate-induced SR Ca(2+) release, and enhanced by ACh. Preexposure to 1 or 2 minimum alveolar concentration halothane completely inhibited Ca(2+) influx when extracellular Ca(2+) was reintroduced, whereas isoflurane and sevoflurane produced less inhibition. Only halothane and isoflurane inhibited ACh-induced augmentation of Ca(2+) influx. CONCLUSION: Volatile anesthetics inhibit a Ni/La-sensitive store-operated Ca(2+) influx mechanism in porcine ASM cells, which likely helps maintain anesthetic-induced bronchodilation.     

The effects of ethanol on CA(2+) sensitivity in airway smooth muscle

Halothane and other volatile anesthetics relax air-way smooth muscle (ASM) in part by decreasing the amount of force produced for a given intracellular Ca(2+) concentration (the Ca(2+) sensitivity) during muscarinic receptor stimulation. To determine whether this is a unique property of the volatile anesthetics, we tested the hypothesis that ethanol, another compound with anesthetic properties, also inhibits calcium sensitization induced by muscarinic stimulation of ASM. A beta-escin permeabilized canine tracheal smooth muscle preparation was used. Ethanol was applied to permeabilized muscles stimulated with calcium in either the absence or presence of acetylcholine. In intact ASM, ethanol produced incomplete relaxation (approximately 40%) at concentrations up to 300 mM. Ethanol significantly increased Ca(2+) sensitivity both in the presence and the absence of muscarinic receptor stimulation. Although ethanol did not affect regulatory myosin light chain (rMLC) phosphorylation during stimulation with Ca(2+) alone, it decreased rMLC phosphorylation by Ca(2+) during muscarinic receptor stimulation. Ethanol, like volatile anesthetics, inhibits increases in rMLC phosphorylation produced by muscarinic receptor stimulation at constant [Ca(2+)](i). However, despite this inhibition, the net effect of ethanol is to increase Ca(2+) sensitivity (defined as the force maintained for a given [Ca(2+)](i)) by a mechanism that is independent of changes in rMLC phosphorylation. IMPLICATIONS: In permeabilized airway smooth muscle, ethanol, like volatile anesthetics, inhibits increases in regulatory protein phosphorylation caused by stimulation of the muscle when intracellular calcium concentration is constant. However, unlike volatile anesthetics, ethanol causes a net increase in force through a process not dependent on protein phosphorylation, an action favoring bronchoconstriction.     

Thiopental induces contraction of rat aortic smooth muscle through Ca(2+) release from the sarcoplasmic reticulum

Little is known about the mechanism of thiopental-induced contraction in vascular smooth muscle. This study aimed to clarify this question by conducting isometric tension experiments and (45)Ca(2+) flux measurements in endothelium-denuded rat aortic rings. Thiopental induced a concentration-dependent contraction under basal tension. This contraction was enhanced when rings were precontracted with phenylephrine in the presence of verapamil. In Ca(2+)-free solution, thiopental-induced contraction was reduced but not abolished with high concentrations. Ca(2+) store depletion with a maximum dose of caffeine in Ca(2+)-free solution further reduced the contraction by subsequent thiopental. Ca(2+) store depletion with thapsigargin completely abolished contraction by thiopental. (45)Ca(2+) influx experiment in the presence of verapamil showed that thiopental could not induce any Ca(2+) influx with or without phenylephrine prestimulation. The (45)Ca(2+) efflux experiment showed more evidence of thiopental-induced Ca(2+) release, which was abolished by thapsigargin. In conclusion, thiopental induces contraction in rat aortic smooth muscle by releasing Ca(2+) from the sarcoplasmic reticulum without Ca(2+) influx. IMPLICATIONS: This is the first study providing evidence that thiopental-induced vascular contraction is caused by Ca(2+) release from the sarcoplasmic reticulum of the smooth muscle.     

Isoflurane alters angiotensin II-induced Ca2+ mobilization in aortic smooth muscle cells from hypertensive rats: implication of cytoskeleton

BACKGROUND: Angiotensin II (AngII) is a potent vasoconstrictor involved in the short-term control of arterial blood pressure. Isoflurane was reported to decrease vascular tone through an alteration of vascular smooth muscle cell vasomotor response to several agonists, but its effect on AngII signaling is not known. On the other hand, vascular response to AngII is altered in hypertension. In this study, the authors tested the hypothesis that (1) isoflurane alters AngII-induced intracellular Ca mobilization in aortic vascular smooth muscle cell from Wistar Kyoto and spontaneously hypertensive rats, and (2) this effect could be associated with an alteration of the organization of microtubular network, reported to be involved in AngII signaling. METHODS: The effect of 0.5-3% isoflurane was studied (1) on AngII (10 m)-induced intracellular Ca mobilization, intracellular Ca release from internal stores, and Ca influx in Fura-2 loaded cultured aortic vascular smooth muscle cell isolated from 6-week-old Wistar Kyoto and spontaneously hypertensive rats, using fluorescent imaging microscopy; and (2) on the organization of cytoskeletal elements, using immunofluorescence labeling. RESULTS: In both stains, isoflurane decreased in a concentration-dependent manner AngII-induced intracellular Ca mobilization, Ca release from internal stores, and Ca influx through nifedipine-insensitive Ca channels. This effect occurred at a lower concentrations of isoflurane in Wistar Kyoto rats than in spontaneously hypertensive rats. In both strains, the effect of isoflurane on AngII- Ca mobilization was abolished by impairment with nocodazole, vinblastine, or paclitaxel of microtubules polymerization. Isoflurane directly altered tubular network organization in a concentration-dependent and reversible manner. CONCLUSIONS: Isoflurane decreased AngII-induced Ca mobilization at clinically relevant concentrations, suggesting that vascular response to AngII could be altered during isoflurane anesthesia. The hypertensive strain was found less sensitive than the normotensive one. In both strains, the isoflurane effect was associated with a microtubular network interaction.     

Mechanisms of isoflurane-mediated hyperpolarization of vascular smooth muscle in chronically hypertensive and normotensive conditions

BACKGROUND: The purpose of this study was to compare the effects of isoflurane on membrane and intracellular mechanisms that regulate vascular smooth muscle (VSM) transmembrane potential (Em; which is related to VSM tone) in the spontaneously hypertensive rat (SHR) model of essential hypertension and its normotensive Wistar-Kyoto (WKY) control. METHODS: Vascular smooth muscle Em values were measured in situ in locally denervated, superfused, intact, small (200-300-microm OD) mesenteric arteries and veins in anesthetized 9-12-week-old SHR and WKY. Effects of 1.0 minimum alveolar concentration (0.60 mM) superfused isoflurane on VSM Em were measured before and during superfusion with specific inhibitors of VSM calcium-activated (KCa) and adenosine triphosphate-regulated (KATP) potassium channels, and with endogenous mediators of vasodilatation (nitric oxide, cyclic guanosine monophosphate, protein kinase G, cyclic adenosine monophosphate, and protein kinase A). RESULTS: Isoflurane significantly hyperpolarized small arteries (5 +/- 3.4 mV) and veins (6 +/- 4.7 mV) (pooled SHR and WKY, mean +/- SD). Inhibition of KCa and KATP channels, cyclic adenosine monophosphate, and protein kinase A, but not nitric oxide, cyclic guanosine monophosphate, and protein kinase G, abolished such hyperpolarization equally in SHR and WKY vessels. CONCLUSIONS: Isoflurane-induced in situ VSM hyperpolarization in denervated, small mesenteric vessels involves a similar activation of KCa and KATP channels and cyclic adenosine monophosphate, but not nitric oxide or cyclic guanosine monophosphate, second messenger pathways in both SHR and WKY. A greater isoflurane-induced VSM hyperpolarization (observed previously in neurally intact SHR vessels) suggests enhanced inhibition of elevated sympathetic neural input as a major mechanism underlying such hyperpolarization (and coupled relaxation) in this neurogenic model of hypertension.     

A comparison of non-invasive methods of blood pressure measurement in normotensive and hypertensive pregnant women

We compared two types of automatic non-invasive blood pressure measuring device with sphygmomanometey in 47 normotensive and 38 hypertensive women in the third trimester of pregnancy. An automatic oscillometric device (Accutor) and a volume-clamp device (Finapres) significantly underestimated the diastolic pressure as measured by the fourth Korotkoff sound using a Hawksley random zero sphygmomanometer. The mean difference between the sphygmomanometer and Accutor measurement of diastolic blood pressure was +3.1 mmHg in the normotensive women and +8.3 mmHg in the hypertensive women (P = 0.001). The mean difference between the sphygmomanometer and Finapres measurement of diastolic blood pressure was +6.1 mmHg in the normotensive women and +11.5 mmHg in hypertensive women (P = 0.003). The increased use of continuous non-invasive devices to monitor blood pressure in women with hypertension should be accompanied by sound knowledge of their limitations in this group of patients.     

Isoflurane activates PKC and Ca(2+) -calmodulin-dependent protein kinase II via MAP kinase signaling in cultured vascular smooth muscle cells.

BACKGROUND: Protein kinase C (PKC) and Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) have been implicated in isoflurane-increased force in skinned femoral arterial strips. The extracellular signal-regulated kinases (ERK1/2) of mitogen-activated protein kinase have been shown to be target effectors of PKC and CaMKII. This study examined the role of the ERK1/2 signaling pathway in isoflurane activation of PKC and CaMKII using cultured vascular smooth muscle cells. METHODS: Vascular smooth muscle cells were prepared by cell migration from isolated rabbit femoral arterial segments. Growth of passage of vascular smooth muscle cells (80-90% confluence, passage 5-10) was arrested for 48 h before experiments, during which time phorbol 1,3-diaceylester treatment was used to down-regulate PKC. Cells were treated for 30 min with one of the inhibitors of mitogen-activated protein kinase kinase (PD98059), PKC (Go6976 and bisindolylmaleimide), or CaMKII (KN-93 and KN-62) at 10 microm. After administration of isoflurane, vascular smooth muscle cells were frozen rapidly, homogenized, and centrifuged. The homogenates were used for identification of phosphorylated ERK1/2 or for further centrifugation to separate the membrane from the cytosol for identification of PKC isoforms (alpha and epsilon) by Western blotting. RESULTS: Isoflurane increased ERK1/2 phosphorylation in a dose-dependent manner and reached a plateau at 10 min. PD98059 or down-regulated PKC blocked the increase of phosphorylated ERK1/2 levels by isoflurane, and bisindolylmaleimide, KN-93, or KN-62, but not by Go6976 reduced levels of phosphorylated ERK1/2. The membrane fraction of PKC epsilon but not of PKC alpha was increased by isoflurane. CONCLUSIONS: ERK1/2 signaling is downstream of PKC and CaMKII activated by isoflurane in vascular smooth muscle cells.     

Differential effects of propofol, ketamine, and thiopental anaesthesia on the skeletal muscle microcirculation of normotensive and hypertensive rats in vivo

Background. This study utilized the dorsal microcirculatorychamber (DMC) model to determine differential effects of i.v.propofol, ketamine, and thiopental anaesthesia on the skeletalmuscle microcirculation (10–180 µm) of normotensive(Male Wistar Kyoto, WKY) and hypertensive (spontaneously hypertensiveHarlan, SHR) rats, importantly, comparing responses to a consciousbaseline. Methods. Three weeks following implantation of the DMC in WKY(n=8) and SHR (n=6) (130 g) 0.25 ml 100 g^–1 FITC–BSA(i.v.) was administered and the microcirculation viewed usingfluorescent in vivo microscopy for a 30 min baseline (t=0–30min). This was followed by either propofol, thiopental, ketamine,or saline (i.v. bolus induction over 5 min (t=30–35 min)),then maintenance step-up infusion for 60 min (t=45–105min), so that animals received all four agents 1 week apart(56 experiments). Results. Dilation of A3 arterioles (15–30 µm) andV3 venules (20–40 µm) with propofol was greaterin SHR (t=95 min, A3 36.7 (12)%, V3 15.5 (2.3)%) than WKY (t=95min, A3 19.4 (7.4)%, V3 8.0 (2.3)%) (P<0.05). Constrictionof A3 with ketamine was greater in SHR (t=95 min, A3 –29.1(6.4)%) than WKY (A3 –17.5 (8.8)%) (P<0.05). This wasaccompanied by hypotension with propofol in SHR (–32%decrease in systolic arterial pressure), but not WKY (–6%)and hypertension with ketamine in WKY (–15%) and SHR (–24%)(P<0.05). During thiopental anaesthesia there was dilationof A1 (80–180 µm), A3, and V3 in WKY (P<0.05).Conversely, in SHR dilation of venules (29.2 (8.7)%) was accompaniedby constriction of A1 and A3 (t=95 min, A1 –25.1 (5.9)%,A3 –45.2 (3.1)%) (P<0.05). Conclusion. Within the skeletal muscle microcirculation of hypertensiverats there is enhanced dilation with propofol and constrictionwith ketamine, associated with exaggerated changes in arterialpressure. Thus, dysfunctional control mechanisms at the levelof the microcirculation alter responses to anaesthesia duringhypertension.      

Effects of ketamine on voltage-dependent Ca2+ current in single smooth muscle cells from rabbit portal vein

The effects of ketamine on membrane potentials and voltage-dependent Ca2+ current were studied in dispersed single smooth muscle cells from rabbit portal vein. The resting membrane potential (-56.2 +/- 1.5mV) was not affected by ketamine (10(-5)-10(-3)M). The duration and the amplitude of the action potential evoked by intracellular stimulation were significantly decreased by ketamine in the concentrations of 10(-4)M and 10(-3)M. Voltage-gated Ca2+ current in single smooth muscle cells was apparently decreased by ketamine at concentrations of between 10(-5)M to 10(-3)M. The activation threshold of Ca2+ current (approx. -30mV) was also decreased by ketamine. Therefore, these results suggest that inhibition of the contractile response by ketamine in vascular smooth muscle from rabbit portal vein may be attributable to the inhibition of the Ca2+ current.     

Differential effects of desflurane and halothane on peripheral airway smooth muscle

Volatile anaesthetics have been shown to have direct relaxant effects on airway smooth muscle. We have examined the effects of 0.9, 1.9, and 2.8 dog MAC of desflurane and halothane on isolated proximal and distal canine airways precontracted with acetylcholine. The proximal and distal airway smooth muscle relaxed with increasing concentration of each anaesthetic in a dose-related manner. Desflurane had a greater relaxant effect than halothane on the proximal airway only at 2.8 MAC. Desflurane relaxed the distal airway to a greater extent than halothane at 1.9 and 2.8 MAC. The distal airway smooth muscle was more sensitive to volatile anaesthetics than the proximal airway smooth muscle with either halothane or desflurane at all concentrations tested. This effect may be a result of differences in cartilage content, myosin content, epithelium-dependent effects, receptor density, myofilament sensitivity to Ca2+, sarcoplasmic reticulum Ca2+ control, or ionic fluxes in the proximal airway compared with the distal airway. The increased sensitivity of airway smooth muscle to desflurane compared with halothane is not known but may be related to possible differences in the effects of Ca2+ homeostasis.      

Determination of Ca2+-release from the isolated smooth muscle cells in suspension from the guinea-pig ileum

Collagenase-dispersed cells from the guinea-pig ileum were prepared and Ca2+ release into Ca2+-depleted solution from the isolated single cells obtained by the centrifugation of the dispersed cells on isotonic sucrose solution was determined with a Ca2+-selective electrode. A technique employing an isotonic sucrose solution for washing isolated cells permitted removal of contaminating extracellular fluids and obtaining the isolated cells with minimum loss of cellular Ca2+. The release of Ca2+ from the dispersed cells consisted of at least two phases. The Ca2+ release into an isotonic sucrose-Tris solution (ISTS) was significantly reduced and the early phase of the Ca2+ release disappeared. At 16 degrees C, Ca2+ release depended on the composition of the bathing solution in a Ca2+-free salt solution, the later phase of Ca2+-release was abolished whereas in ISTS both phases disappeared. Furthermore, Ca2+ release was significantly reduced after the treatment of the dispersed cells with disperse (1,500 units/ml) for 10 min. These results show that Ca2+ release into Ca2+-depleted solution from the isolated ileal cells with minimum loss of cellular Ca2+ show a biphasic curve and suggest that Ca2+ sources responsible for these phases may be of distinct origin.     

Interaction of arginine vasopressin and angiotensin II on Ca2+ in vascular smooth muscle cells

The non-osmotic release of arginine vasopressin (AVP) is associated with the concomitant activation of the renin-angiotensin and sympathetic nervous systems. In vivo studies suggest that a positive interaction may occur between AVP and angiotensin II (Ang II), and other Ca2+ mobilizing hormones. In the present study, the cellular mechanisms of this interaction between AVP and Ang II in vascular smooth muscle cell (VSMC) were examined. These results support the existence of a positive interaction between AVP and Ang II on Ca2+ mobilization in VSMC. In fact, the challenge of VSMC with combined AVP and Ang II, in a range from 5 x 10(-11) to 10(-8) M, enhanced cytosolic free Ca2+ ([Ca2+]i) and 45Ca2+ efflux in a more than additive manner. This potentiation, which was not dependent of the presence of extracellular calcium, correlated with an increased VSMC shape change. Moreover, the combination of subthreshold doses of AVP and Ang II (5 x 10(-11) M), which do not release Ca2+ alone, evoked a Ca2+ mobilizing response. A subthreshold dose of Ang II also shifted to the left the concentration-response curve of the AVP-mediated 45Ca2+ efflux. Since there were no changes in receptor binding of either hormone by the other hormone and the interaction of the two hormones on the production of inositol phosphatides was additive, the AVP and AII positive interaction on Ca2+ mobilization on VSMC may occur at the level of the intracellular Ca2(+)-releasing mechanism itself. Such an interaction can occur at hormone concentrations below the Ca2+ release threshold and may explain an increased functional response to the combination of pressor hormones compared to that of each hormone alone.     

Spontaneous Ca2+ activated Cl- currents in isolated urethral smooth muscle cells

PURPOSE: We identified and characterized the membrane currents underlying spontaneous transient depolarization in the urethra. MATERIALS AND METHODS: Myocytes were isolated from sheep urethra by enzymatic digestion and studied by the amphotericin B patch clamp method. RESULTS: Just more than 10% of cells had spontaneous transient inward currents when maintained at -60 mV. Mean amplitude plus or minus standard error of mean of the spontaneous transient inward currents was 102 +/- 35 pA. and mean frequency was 17 +/- 3 minutes-1 in 18 preparations. Within each cell currents sometimes consisted of up to 3 phases but in 16 of 18 cells monophasic spontaneous transient inward currents were also identified. These currents decayed relatively slowly with a mean time constant of 570 +/- 97 ms. Spontaneous transient inward currents were identified as Ca2+ activated Cl- currents because they reversed near the calculated Nernst potential for chloride ions. They were blocked by the Cl- channel blockers 100 microM. niflumic acid and 1 mM. anthracene-9-carboxylic acid as well as in Ca2+-free solution, 10 mM. caffeine and 30 microM. ryanodine. The latter results suggest that spontaneous transient inward currents require intact intracellular Ca2+ stores. Amplitude and frequency were unaffected by 10 microM. nifedipine but were reduced by the nonspecific Ca2+ entry blockers 10 microM. SKF 96365 and 1 mM. La3+. We interpret these results as indicating that the Ca2+ stores underlying the spontaneous transient inward currents may refill by plasmalemmal Ca2+ channels that differ from L-type channels. CONCLUSIONS: Urethral cells fire large spontaneous transient inward currents, mediated by Ca2+ activated Cl- channels, which are adequate to account for the spontaneous transient depolarizations seen in whole urethral tissue.     

Beta2-microglobulin and hypertensive complications in pregnant women at risk

BACKGROUND AND AIM: Beta2-microglobulin (beta2-m) is a polypeptide, which is freely filtered through the glomerular basement membrane and absorbed almost entirely by the proximal tubular cells. Preeclampsia, a common complication of pregnancy, is characterized by pathological renal changes, mainly glomerular lesions. The aim of the present study was to investigate whether serum beta2-m measured in the early stages of pregnancy could be used as a marker to predict hypertensive complications in women at increased risk. PATIENTS AND METHODS: Serum beta2-m concentrations were prospectively measured in 75 pregnant women with history of chronic hypertension, chronic renal disease, chronic vascular disease or preeclampsia and compared with those in 16 healthy pregnant women. RESULTS: Of the 75 women in the study group, 10 (13%) developed preeclampsia and 20 (26%) had other complications, such as intrauterine growth restriction (n = 8), fetal or neonatal loss (n = 9) and delivery before 30 weeks of gestation (n = 8). Gestational age at delivery, birth weight and cesarean section rate were significantly worse in the patients with complications than in those without and in the healthy controls. No significant difference was detected in early serum beta2-m concentrations between the women who later developed preeclampsia or other complications and those who did not. There was a significant positive correlation of beta2-m concentrations with serum creatinine level (R2 = 0.394, p < 0.001), but not with gestational week at blood collection. CONCLUSION: Serum beta2-m concentrations are not predictive of the development of preeclampsia or other complications in woman at risk.     

Ryanodine receptor and capacitative Ca2+ entry in fresh preglomerular vascular smooth muscle cells

BACKGROUND: A multiplicity of hormonal, neural, and paracrine factors regulates preglomerular arterial tone by stimulating calcium entry or mobilization. We have previously provided evidence for capacitative (store-operated) Ca2+ entry in fresh renal vascular smooth muscle cells (VSMCs). Ryanodine-sensitive receptors (RyRs) have recently been identified in a variety of nonrenal vascular beds. METHODS: We isolated fresh rat preglomerular VSMCs with a magnetized microsphere/sieving technique; cytosolic Ca2+ ([Ca2+]i) was measured with fura-2 ratiometric fluorescence. RESULTS: Ryanodine (3 micromol/L) increased [Ca2+]i from 79 to 138 nmol/L (P = 0.01). Nifedipine (Nif), given before or after ryanodine, was without effect. The addition of calcium (1 mmol/L) to VSMCs in calcium-free buffer did not alter resting [Ca2+]i. In Ca-free buffer containing Nif, [Ca2+]i rose from 61 to 88 nmol/L after the addition of the Ca2+-ATPase inhibitor cyclopiazonic acid and to 159 nmol/L after the addition of Ca2+ (1 mmol/L). Mn2+ quenched the Ca/fura signal, confirming divalent cation entry. In Ca-free buffer with Nif, [Ca2+]i increased from 80 to 94 nmol/L with the addition of ryanodine and further to 166 nmol/L after the addition of Ca2+ (1 mmol/L). Mn2+ quenching was again shown. Thus, emptying of the sarcoplasmic reticulum (SR) with ryanodine stimulated capacitative Ca2+ entry. CONCLUSION: Preglomerular VSMCs have functional RyR, and a capacitative (store-operated) entry mechanism is activated by the depletion of SR Ca2+ with ryanodine, as is the case with inhibitors of SR Ca2+-ATPase.