Postnatal maturation attenuates pressure-evoked myogenic tone and stretch-induced increases in Ca2+ in rat cerebral arteries

Although postnatal maturation potently modulates agonist-induced cerebrovascular contractility, its effects on the mechanisms mediating cerebrovascular myogenic tone remain poorly understood. Because the regulation of calcium influx and myofilament calcium sensitivity change markedly during early postnatal life, the present study tested the general hypothesis that early postnatal maturation increases the pressure sensitivity of cerebrovascular myogenic tone via age-dependent enhancement of pressure-induced calcium mobilization and myofilament calcium sensitivity. Pressure-induced myogenic tone and changes in artery wall intracellular calcium concentrations ([Ca(2+)](i)) were measured simultaneously in endothelium-denuded, fura-2-loaded middle cerebral arteries (MCA) from pup [postnatal day 14 (P14)] and adult (6-mo-old) Sprague-Dawley rats. Increases in pressure from 20 to 80 mmHg enhanced myogenic tone in MCA from both pups and adults although the normalized magnitudes of these increases were significantly greater in pup than adult MCA. At each pressure step, vascular wall [Ca(2+)](i) was also significantly greater in pup than in adult MCA. Nifedipine significantly attenuated pressure-evoked constrictions in pup MCA and essentially eliminated all responses to pressure in the adult MCA. Both pup and adult MCA exhibited pressure-dependent increases in calcium sensitivity, as estimated by changes in the ratio of pressure-induced myogenic tone to wall [Ca(2+)](i). However, there were no differences in the magnitudes of these increases between pup and adult MCA. The results support the view that regardless of postnatal age, changes in both calcium influx and myofilament calcium sensitivity contribute to the regulation of cerebral artery myogenic tone. The greater cerebral myogenic response in P14 compared with adult MCA appears to be due to greater pressure-induced increases in [Ca(2+)](i), rather than enhanced augmentation of myofilament calcium sensitivity.     

Action of the Calcium Antagonists Cocaine and Ethanol on Contraction and Potassium Efflux of Smooth Muscle

Isolated longitudinal smooth muscle from guinea pig ileum exposed to a high potassium depolarizing medium exhibited a sustained increase in muscle tone and an increase in potassium efflux. When the concentration of calcium ion in the medium was elevated the increase in muscle tone was enhanced, but the change in potassium efflux was reduced slightly. Lowering the calcium concentration diminished the increase in muscle tone. Both cocaine and ethanol completely inhibited the sustained contraction of potassium-depolarized fibers. Addition of excess calcium ion reversed these inhibitions. Cocaine acted primarily like a competitive antagonist; and ethanol, like an indirect antagonist of calcium, ion. Under certain conditions acetylcholine potentiated the reversal by calcium ion of the drug-induced inhibitions. The two inhibitory drugs had dissimilar effects on potassium efflux from smooth muscle fibers immersed in Tyrode solution. Cocaine depressed and ethanol enhanced this membrane process. However, the increase in potassium efflux induced by acetylcholine was inhibited by ethanol. This inhibition also was reversed by increasing the concentration of calcium ion in the medium. The data suggested that calcium activates and cocaine and ethanol inhibit a cellular reaction which occurs beyond the point of membrane depolarization and is essential for smooth muscle contraction. Furthermore, calcium serves to depress membrane excitability, but appears to have a specific stimulatory role in the acetylcholine-induced increase in potassium efflux from longitudinal fibers.     

Expression of myogenic constrictor tone in the aorta of hypertensive rats

We investigated the effect of intraluminal pressure or stretch on the development of tone in the descending thoracic aorta from rats with aortic coarctation-induced hypertension of 7-14 days duration. Increments of pressure >100 mmHg decreased the diameter of thoracic aortas from hypertensive but not from normotensive rats. The pressure-induced constriction was not demonstrable in vessels superfused with calcium-free buffer. Stretched rings of aorta from hypertensive rats exhibited a calcium-dependent constrictor tone accompanied by elevated calcium influx that varied in relation to the degree of stretch. Blockers of L-type calcium channels and inhibitors of protein kinase C reduced both basal tone and calcium influx in aortic rings of hypertensive rats. Hence, the thoracic aorta of hypertensive rats expresses a pressure- and stretch-activated constrictor mechanism that relies on increased calcium influx through L-type calcium channels via a protein kinase C-regulated pathway. The expression of such a constrictor mechanism is suggestive of acquired myogenic behavior.     

Relative contribution of Rho kinase and protein kinase C to myogenic tone in rat cerebral arteries in hypertension

Arterial smooth muscle constriction in response to pressure, i.e., myogenic tone, may involve calcium-dependent and calcium-sensitization mechanisms. Calcium sensitization in vascular smooth muscle is regulated by kinases such as PKC and Rho kinase, and activity of these kinases is known to be altered in cardiovascular disorders. In the present study, we evaluated the relative contribution of PKC and Rho kinase to myogenic tone in cerebral arteries in hypertension. Myogenic tone and arterial wall calcium in Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) were measured simultaneously, and the effect of PKC and Rho kinase inhibitors on myogenic tone was evaluated. SHR arteries showed significantly greater myogenic tone than WKY arteries. Pressure/wall tension-arterial wall calcium curves showed a hyperbolic relation in WKY rats, but the curves for SHR arteries were parabolic. Myogenic tone was decreased by the Rho kinase inhibitors Y-27632 and HA-1077, with a significantly greater effect in SHR than in WKY arteries. Reduction in myogenic tone produced by the PKC inhibitor bisindolylmaleimide I in WKY and SHR arteries was significantly less than that produced by Rho kinase inhibition. The pressure-dependent increase in myogenic tone was significantly decreased by Y-27632, and the decrease was markedly greater than that produced by bisindolylmaleimide I in SHR arteries. In WKY arteries, the pressure-dependent increase in myogenic tone was decreased to a similar extent by Y-27632 and bisindolylmaleimide I. These results suggest greater myogenic tone with increased calcium sensitization in SHR arteries, largely because of Rho kinase activation, with a minor contribution of PKC activation.     

Rho kinase inhibition partly weakens myogenic reactivity in rat small arteries by changing calcium sensitivity

The hypothesis that Rho kinase is involved in myogenic reactivity was investigated in pressurized rat tail small arteries using videomicroscopic diameter determination and calcium fluorimetry. The potent Rho kinase inhibitor Y-27632 reversibly increased vessel diameter at 80 mmHg without changing the intracellular calcium concentration ([Ca](i)) shifting the relationship between diameter change and [Ca](i) to higher calcium levels. Neither endothelium removal nor inhibition of neural transmission affected the Y-27632-induced effect. Y-27632 at 3 x 10(-6) mol/l attenuated the myogenic response in the pressure range from 10 to 120 mmHg, shifting the relationship between vessel tone and [Ca](i) to higher calcium levels. In addition, the Y-27632-induced shift of the relationship between vessel tone and [Ca](i) was larger at 80 than at 10 mmHg. These results suggest that smooth muscle cell Rho kinase in rat tail small arteries 1) is in an active state partly determining the level of the myogenic tone, and 2) alters the strength of the myogenic response by changing calcium sensitivity, probably caused by the pressure-induced activation of the kinase.     

CaMKIIT287 and T305 regulate history-dependent increases in alpha agonist-induced vascular tone.

CaMKII is a calcium and calmodulin-activated kinase that has been shown to regulate learning and memory in the brain, and contractility in blood vessels. Following Ca activation, CaMKII autophosphorylates, gaining a calcium-independent autonomous activity that reflects a molecular memory of having previously come into contact with calcium. The present study addresses whether the molecular memory properties of CaMKII are involved in the modulation of sustained vascular tone. We demonstrate a history-dependence of alpha agonist-induced vascular tone and show that CaMKII activation in vascular cells is also history dependent. Autophosphorylation of Thr287, which is classically associated with autonomous activity, does not persist during tone maintenance after transient increases in intracellular calcium levels. However, we have found that another site, Thr305, known from in vitro studies to be inhibitory, is regulated by alpha agonists in that the inhibitory action is removed, thus leading to a delayed reactivation of CaMKII as measured by Thr287 phosphorylation. By the use of a small molecule CaMKII inhibitor (KN93) as well as a decoy peptide (autoinhibitory peptide; AIP) we show a cause and effect relationship between CaMKII reactivation and sustained vascular tone maintenance. Thus, it appears that a complex interplay between the regulation of Thr305 and Thr287 provides a novel mechanism by which a history-dependence is developed and contributes to a new facet of molecular memory for CaMKII of relevance to vascular tone maintenance.     

Myogenic contractility is more dependent on myofilament calcium sensitization in term fetal than adult ovine cerebral arteries

Regulation of cytosolic calcium and myofilament calcium sensitivity varies considerably with postnatal age in cerebral arteries. Because these mechanisms also govern myogenic tone, the present study used graded stretch to examine the hypothesis that myogenic tone is less dependent on calcium influx and more dependent on myofilament calcium sensitization in term fetal compared with adult cerebral arteries. Term fetal and adult posterior communicating cerebral arteries exhibited similar myogenic responses, with peak tensions averaging 24 and 26% of maximum contractile force produced in any given tissue in response to an isotonic Krebs buffer containing 122 mM K(+) (K(max)) at optimum stretch ratios (working diameter/unstressed diameter) of 2.19 and 2.23, respectively. Graded stretch increased cytosolic Ca(2+) concentration at stretch ratios >2.0 in adult arteries, but increased Ca(2+) concentration only at stretch ratios >2.3 in fetal arteries. In permeabilized arteries, myogenic tone peaked at a stretch ratio of 2.1 in both fetal and adult arteries. The fetal %K(max) values at peak myogenic tone were not significantly different at either pCa 7.0 (23%) or pCa 5.5 (25%) but were significantly less at pCa 8.0 (8.4 +/- 2.3%). Conversely, adult %K(max) values at peak myogenic tone were significantly less at both pCa 8.0 (10.4 +/- 1.8%) and pCa 7.0 (16%) than at pCa 5.5 (27%). The maximal extents of stretch-induced increases in myosin light chain phosphorylation in intact fetal (20%) and adult (17%) arteries were similar. The data demonstrate that the cerebrovascular myogenic response is highly conserved during postnatal maturation but is mediated differently in fetal and adult cerebral arteries.     

Parathyroid hormone causes a transient rise in intracellular ionized calcium in vascular smooth muscle cells

The effect of parathyroid hormone on intracellular calcium concentration in vascular smooth muscle cells in culture was studied. Human PTH 1-34 (hPTH (1-34)) caused a transient rise in intracellular calcium in a dose-dependent manner at physiological concentrations. The effect of PTH was mimicked by dibutyryl cyclic AMP and inhibited by a PTH receptor antagonist. The effect of PTH was increased in parallel with extracellular calcium concentration and a sustained response was observed when extracellular calcium was 2 mM or higher. The PTH action was blocked by nisoldipin, a calcium antagonist, but not by ouabain, a Na, K-ATPase inhibitor. These data indicate that PTH increases intracellular calcium through its receptor via opening calcium channels. A possible role of this effect in the regulation of vascular tone is also discussed.     

Inositol 1,4,5-trisphosphate receptor subtypes are differentially distributed between smooth muscle and endothelial layers of rat arteries

In blood vessels, the ability to control vascular tone depends on extracellular calcium entry and the release of calcium from inositol 1,4,5-trisphosphate receptor (IP3R)-gated stores located in both the endothelial and smooth muscle cells of the vascular wall. Therefore, we examined mRNA expression and protein distribution of IP3R subtypes in intact aorta, basilar and mesenteric arteries of the rat. IP3R1 mRNA was predominantly expressed in all three arteries. Immunohistochemistry showed that IP3R1 was present in both the muscle and endothelial cell layers, while IP3R2 and IP3R3 were largely restricted to the endothelium. Weak expression of IP3R2 was observed in the smooth muscle of the basilar artery. Co-localisation studies of IP3R subtypes with known cellular elements showed no association of any of the three subtypes with the endothelial cell plasma membrane, but a close association between the subtypes and actin filaments was observed in all cell layers. IP3R2 was found to be present near the endothelial cell nucleus. We are the first to demonstrate differential IP3R subtype distribution between the cell layers of the intact vascular wall and hypothesise that this may underlie the diversity of IP3R-dependent responses, such as vasoconstriction, vasodilation and vasomotion, displayed by arteries.     

Maturation depresses cGMP-mediated decreases in [Ca2+]i and Ca2+ sensitivity in ovine cranial arteries

Because cerebrovascular cGMP levels vary significantly during maturation, we examined the hypothesis that the ability of cGMP to relax cerebral arteries also changes during maturation. In concentration-response experiments, potassium-induced tone in basilar arteries was significantly more sensitive to a nonmetabolizable cell-permeant cGMP analogue 8-(p-chlorophenylthio)-cGMP (8-pCPT-cGMP) in term fetal [-log one-half maximal concentration (EC(50)) = 4.4 +/- 0.1 M] than in adult (-log EC(50) = 4.0 +/- 0.1 M) ovine basilar arteries. Serotonin-induced tone also revealed significantly greater sensitivity to the cGMP analogue in fetal (-log EC(50) = 4.9 +/- 0.1 M) than in adult (-log EC(50) = 4.7 +/- 0.1 M) basilars. In fura 2-loaded preparations, 8-pCPT-cGMP had no significant effect on cytosolic calcium concentrations in potassium-contracted arteries but at 6 microM significantly reduced calcium only in fetal basilars (Delta = 33 +/- 8%). Higher 8-pCPT-cGMP concentrations reduced cytosolic calcium in both fetal and adult basilars. Similarly, in both potassium- and 5-hydroxytryptamine (5-HT)-contracted preparations, low concentrations of 8-pCPT-cGMP reduced myofilament calcium sensitivity only in fetal basilars (Delta = 29 +/- 6 and Delta = 42 +/- 10%, respectively), whereas higher concentrations reduced calcium sensitivity in both fetal and adult arteries. In beta-escin-permeabilized arteries, equivalent reductions in basal and agonist-enhanced myofilament calcium sensitivity were produced by much lower 8-pCPT-cGMP concentrations in fetal (172 and 61 microM, respectively) than in adult (410 and 231 microM, respectively) basilars. The mechanisms mediating cGMP-induced vasorelaxation appear similar in fetal and adult arteries, with the exception that they are much more sensitive to cGMP in fetal than adult arteries. These age-related differences in the sensitivity of cytosolic calcium concentration, basal, and agonist-enhanced myofilament calcium sensitivity to cGMP can easily explain why both potassium- and 5-HT-induced tone are more sensitive to cGMP in fetal than adult cerebral arteries.     

Effect of small changes in extracellular magnesium concentration on the tone of feline mesenteric arteries: involvement of endothelium.

The aim of this study was to investigate the effect of small alterations in extracellular magnesium concentration on the tone of feline mesenteric arteries and to examine the role of endothelium in these responses. We measured isometrical tension of isolated arterial rings, placed between two stainless steel wires in a tissue chamber containing Krebs-Henseleit solution, aerated with a gas mixture containing 95% O2 and 5% CO2 at 37 degrees C. After precontraction with noradrenaline, a decrease of extracellular magnesium concentration from 1.2 mM to 1.0 and 0.8 mM resulted in sustained relaxations, whereas the elevation of extracellular magnesium from 0.8 mM to 1.2 mM caused an increase in vascular tone when endothelium was intact. The magnesium-withdrawal related dilations were absent in endothelium-denuded vessels and were inhibited by oxyhemoglobin (5 x 10(-6) M) and methylene blue (10(-5) M), suggesting the involvement of endothelium-derived relaxing factor in this vascular response. Nifedipine (5 x 10(-7) M) or dichlorobenzamil (3 x 10(-5) M), however, did not affect the magnesium-deficiency related relaxations. Therefore, in this vascular action of magnesium, nifedipine-sensitive calcium channels or the sodium- calcium antiport system are not involved. We conclude that small alterations in extracellular magnesium concentration, possibly within the physiological range, are able to modify the basal formation and release of EDRF, and thus alter arterial smooth muscle tone in this vascular bed. This endothelium- and magnesium-dependent system appears to be more sensitive than the direct smooth muscle actions of magnesium. The possible physiological and pathophysiological consequences of these observations are discussed.     

Mechanisms of spontaneous relaxation of smooth muscle (guinea pig taenia coli) depolarized in a hyperkalemic medium]

Experiments were performed on isolated strips of guinea pig taenia coli by the double sucrose-gap method. The artificial node was depolarized with potassium solution (from 120 to 167.7 mM KCl). When the bathing solution contained 0.4 mM Ca and the temperature was equal to 25 degrees C then potassium contracture was followed by fast relaxation. The muscular tone changed slightly during rectangular pulse of hyperpolarizing current, after switching off the current muscle generated a transient contractile response. The amplitude of such off-responses increased in some range with increasing in strength and duration of conditioning current. Treatment of muscle with compound D-600 resulted in a reduction of muscular tone and elimination of off-responses. The addition of Na ions to potassium solution (substitution of 47.7 mM KCl with the same quantity of NaCl) reduced muscular tone and enhanced the relaxation after off-responses. In sodium-free potassium solution each off-response was followed by increasing muscular tone but when the bathing solution contained Na ions this increase of the tone was not observed. The data obtained strongly suggest that the spontaneous relaxation of smooth muscle which was contracted in K-solution resulted from: 1) inactivation of calcium channels of surface membrane, 2) sequastration of Ca ions by intracellular storange sites, 3) extrusion of Ca in extracellular space (in part by means of Na-Ca exchange diffusion).     

Vascular adaptations to pregnancy in mice: effects on myogenic tone

The mechanisms underlying vascular adaptations in pregnancy remain to be fully elucidated. One of the contributory mechanisms for reduced vascular tone may be a reduction of myogenic tone. Myogenic tone was assessed as the difference between internal diameter in the presence and absence of external calcium at different intramural pressure steps (60-100 mmHg). Myogenic responses were reduced in resistance-sized mesenteric and main uterine arteries in late pregnant compared with nonpregnant C57BL/6J mice. In vessels from pregnant, but not nonpregnant mice, the myogenic response was enhanced by preincubation with nitric oxide (NO) synthase inhibitor N(G)-nitro-l-arginine methyl ester, was further elevated by the gap junction inhibitor 18-alpha glycyrrhetinic acid, but was unaltered by the prostaglandin H synthase inhibitor meclofenamate. Endothelium removal enhanced myogenic tone only in the vessels from pregnant animals, thus confirming the role of the endothelium in modulating myogenic tone in pregnancy. These results suggest that endothelium-derived NO as well as gap junction communications modulate myogenic tone in mouse pregnancy.     

Antioxidant effects and the therapeutic mode of action of calcium channel blockers in hypertension and atherosclerosis

Drugs currently known as calcium channel blockers (CCB) were initially called calcium antagonists because of their ability to inhibit calcium-evoked contractions in depolarized smooth muscles. Blocking the entry of calcium reduces the active tone of vascular smooth muscle and produces vasodilatation. This pharmacological property has been the basis for the use of CCBs in the management of hypertension and coronary heart disease. A major question is whether drugs reducing blood pressure have other effects that help prevent the main complications of hypertension, such as atherosclerosis, stroke, peripheral arterial disease, heart failure and end-state renal disease. Experimental studies that focus on this question are reviewed in the present paper.     

Paradoxical effects of temperature on vascular tone

Temperature may have significant influence on vascular tone in such cases as organ preservation, coronary bypass surgery, and extracorporeal circulation. The aim of this research was to study the direct effect of temperature variation on vascular tone in an attempt to elucidate the mechanisms involved. In a first series of experiments, the isometric tension of two different vessels (rat thoracic aorta and pig renal branch artery) was studied at different temperatures. To study the role of calcium in this response, a second series of experiments was performed. In this the vessels were incubated with the intracellular chelator BAPTA/AM. Further experiments were performed to test the effect of cold storage. Our results show that changes in temperature lead to different results in pig renal artery and rat aorta. A decrease in temperature induced a highly reproducible relaxation in rat aorta, whereas pig renal artery presented cooling-induced contraction. Moreover, whereas calcium depletion failed to inhibit cooling-induced relaxation in rat aorta, it did not provoke cooling-induced contraction in pig renal artery. Similar responses were obtained with cold storage and calcium depletion. We intend to demonstrate that, just as the effect of temperature variation on pig renal artery is due to a metabolic mechanism, its effect on rat aorta may be due to structural factors. This hypothesis is supported by the result of histological studies which demonstrate a higher proportion of elastin fibres in rat aorta than in pig renal artery.     

Possible mechanisms of oxygen sensing in the pulmonary circulation.

Oxygen tension is known to control the pulmonary vascular tone. We reviewed three hypotheses that try to explain the mechanism whereby hypoxia is sensed in the lung tissue. The first hypothesis concerns the role of the oxygen binding hemoprotein cytochrome P-450. Studies using various inhibitors and activators of cytochrome P-450 show that this enzyme affects pulmonary vascular tone. The data are, however, contradictory. The second hypothesis postulates that hypoxia reduces the synthesis of vasodilator oxygen radicals in the lung. This hypothesis is quite well supported by experimental data. The third hypothesis, similarly widely documented, states that slowing of the respiratory chain and altered cellular energetics is crucial for sensing of hypoxia. In this case, however, it is not exactly clear how changes in cellular energetics are connected with vascular tone. The possibility exists that changes in both the cytochrome P-450 activity and in the rate of electrons flow in the respiratory chain may alter the amount of oxygen radicals in the cells and, similarly as in the "oxygen radicals" hypothesis, govern calcium channels through the control of the redox status of these channels.     

Acidity and species diversity in freshwater crustacean faunas

SUMMARY. On the basis of material from over seventy acidic water bodies in three different areas a relationship is revealed between pH and the species diversity of their crustacean faunas. With increasing acidity species diversity decreases. No such relationship is shown between calcium concentration and species diversity. The very low calcium levels of such waters may, however, play a part in the exclusion of certain species.     

Calcium signal communication in the central nervous system

The communication of calcium signals between cells is known to be operative between neurons where these signals integrate intimately with electrical and chemical signal communication at synapses. Recently, it has become clear that glial cells also exchange calcium signals between each other in cultures and in brain slices. This communication pathway has received utmost attention since it is known that astrocytic calcium signals can be induced by neuronal stimulation and can be communicated back to the neurons to modulate synaptic transmission. In addition to this, cells that are generally not considered as brain cells become progressively incorporated in the picture, as astrocytic calcium signals are reported to be communicated to endothelial cells of the vessel wall and can affect smooth muscle cell tone to influence the vessel diameter and thus blood flow. We review the available evidence for calcium signal communication in the central nervous system, taking into account a basic functional unit -the brain cell tripartite- consisting of neurons, glial cells and vascular cells and with emphasis on glial-vascular calcium signaling aspects.     

Calcium source diversity in feline lower esophageal sphincter circular and sling muscle

Within muscular equivalents of cat lower esophageal sphincter (LES), the circular muscle develops greater spontaneous tone, whereas the sling muscle is more responsive to cholinergic stimulation. Smooth muscle contraction involves a combination of calcium release from stores and of calcium entry via several pathways. We hypothesized that there are differences in the sources of Ca(2+) used for contraction in sling and circular muscles and that these differences could contribute to functional asymmetry observed within LES. Contraction of muscle strips from circular and sling regions of LES was assessed in the presence of TTX. In Ca(2+)-free Krebs, tone was inhibited to a greater degree in circular than sling muscle. L-type Ca(2+) channel blockade with nifedipine or verapamil inhibited tone in LES circular but not sling muscle. Sarcoplasmic reticulum (SR) Ca(2+)-ATPase inhibitor cyclopiazonic acid (CPA) caused greater increase in tone in sling than in circular muscle. The phospholipase C inhibitor U-73122 and the SR inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] receptor blocker 2-aminoethoxydiphenyl borate (2-APB) inhibited tone in circular and sling muscles, demonstrating that continuous release of Ca(2+) from Ins(1,4,5)P(3)-sensitive stores is important in tone generation in both muscles. In Ca(2+)-free Krebs, ACh-induced contractions (AChC) were inhibited to a greater degree in sling than circular muscles. However, nifedipine and verapamil greatly inhibited AChC in the circular but not sling muscle. Depletion of SR Ca(2+) stores with CPA or inhibition of Ins(1,4,5)P(3)-mediated store release with either U-73122 or 2-APB inhibited AChC in both muscles. We demonstrate that LES circular and sling muscles 1) use intracellular and extracellular Ca(2+) sources to different degrees in the generation of spontaneous tone and AChC and 2) use different Ca(2+) entry pathways. These differences hold the potential for selective modulation of LES tone in health and disease.