Lovastatin Induces Relaxation and Inhibits L‐Type Ca2+ Current in the Rat Basilar Artery

Abstract: Statins inhibit cholesterol biosynthesis and protect against ischaemic stroke. It has become increasingly apparent that the beneficial effects of statin therapy may extend beyond lowering of serum cholesterol. The present study was done to explore possible pleiotropic statin effects at the level of the cerebral vascular smooth muscle. Lovastatin, lovastatin acid, simvastatin and pravastatin, were added to segments of the rat basilar artery and effects on contraction and Ca²⁺ handling were examined. Pravastatin had no effect on contraction. Simvastatin, lovastatin, and, to a lesser degree, lovastatin acid, caused relaxation (IC₅₀=0.8, 1.9 and 22 μmol/l) of both intact and denuded arteries precontracted with 5‐HT or high‐K⁺. This effect was not reversed by mevalonate, suggesting that it was not related to cholesterol or isoprenoid metabolism. Relaxation was associated with a reduction of the intracellular Ca²⁺ concentration measured with Fura 2 and with a reduced Mn²⁺ quench rate, suggesting a direct effect on ion channels in the smooth muscle cell membrane. Current measurements in isolated and voltage clamped basilar artery muscle cells demonstrated that both lovastatin and lovastatin acid inhibit L‐type Ca²⁺ current. We propose that lipophilicity is an important factor behind the effects of statins on vascular tone and that Ca²⁺ current inhibition is the likely mechanism of action.     

The involvement of protein kinase C and tyrosine kinase in vanadate-induced contraction

Gastric smooth muscle of cats was used to investigate the involvement of protein kinase in vanadate-induced contraction. Vanadate caused a contraction of cat gastric smooth muscle in a dose-dependent manner. Vanadate-induced contraction was totally inhibited by 2 mM EGTA and 1.5 mM LaCl₃ and significantly inhibited by 10 μM verapamil and 1 μM nifedipine, suggesting that vanadate-induced contraction is dependent on the extracellular Ca²⁺ concentration, and the influx of extracellular Ca²⁺ was mediated through voltage-dependent Ca²⁺ channel. Both protein kinase C inhibitor and tyrosine kinase inhibitor significantly inhibited the vanadate-induced contraction and the combined inhibitory effect of two protein kinase inhibitors was greater than that of each one. But calmodulin antagonists did not have any influence on the vanadate-induced contraction. On the other hand, both forskolin (1 μM) and sodium nitroprusside (1 μM) significantly inhibited vanadate-induced contraction. Therefore, these results suggest that both protein kinase C and tyrosine kinase are involved in the vanadate-induced contraction which required the influx of extracellular Ca²⁺ in cat gastric smooth muscle, and that the contractile mechanism of vanadate may be different from that of agonist binding to its specific receptor.     

Calcium Sensitization Induced by Sodium Fluoride in Permeabilized Rat Mesenteric Arteries

It was hypothesized that NaF induces calcium sensitization in Ca²⁺-controlled solution in permeabilized rat mesenteric arteries. Rat mesenteric arteries were permeabilized with β-escin and subjected to tension measurement. NaF potentiated the concentration-response curves to Ca²⁺ (decreased EC₅₀ and increased E_max). Cumulative addition of NaF (4.0, 8.0 and 16 mM) also increased vascular tension in Ca²⁺-controlled solution at pCa 7.0 or pCa 6.5, but not at pCa 8.0. NaF-induced vasocontraction and GTPγS-induced vasocontraction were not additive. NaF-induced vasocontraction at pCa 7.0 was inhibited by pretreatment with Rho kinase inhibitors H1152 or Y27632 but not with a MLCK inhibitor ML-7 or a PKC inhibitor Ro31-8220. NaF induces calcium sensitization in a Ca²⁺-dependent manner in β-escin-permeabilized rat mesenteric arteries. These results suggest that NaF is an activator of the Rho kinase signaling pathway during vascular contraction.     

Investigation of Mechanisms Involved in (−)‐Borneol‐Induced Vasorelaxant Response on Rat Thoracic Aorta

Abstract: The monoterpene (?)‐borneol is present in essential oils of several medicinal plants. The aim of this study was to evaluate (?)‐borneol effects on rat thoracic aorta artery rings. The cumulative addition of (?)‐borneol (10^?9–3 × 10^?4 M) on a phenylephrine‐induced pre‐contraction (10^?6 M) promoted a vasorelaxant effect in a concentration‐dependent manner and independent of vascular endothelium. A similar effect was obtained on KCl‐induced pre‐contractions (80 mM). (?)‐Borneol (10^?5–3 × 10^?4 M) inhibited contractions induced by cumulative addition of CaCl₂ (10^?6–3 × 10^?2 M) in depolarizing medium without Ca²⁺ in a concentration‐dependent manner. On S‐(?) Bay K 8644‐induced pre‐contractions (10^?7 M), (?)‐borneol did not induce significant changes compared with KCl‐induced pre‐contractions. In a Ca²⁺‐free medium, (?)‐borneol (10^?5, 10^?4 or 10^?3 M) interfered in calcium mobilization from phenylephrine (10^?6 M)‐ or caffeine (20 mM)‐sensitive intracellular stores. The involvement of K⁺ channels was evaluated by tetraethylammonium (3 mM), 4‐aminopyridine (1 mM) and glibenclamide (10^?5 M) pre‐treatment, and (?)‐borneol‐induced vasorelaxation was markedly attenuated. Thus, this vasorelaxant effect can probably be attributed to calcium influx blockade through voltage‐operated calcium channels (Ca_VL), calcium mobilization from intracellular stores and potassium channels activation.     

RELATIVE CONTRIBUTIONS OF EXTRACELLULAR Ca2+ AND Ca2+ STORES TO SMOOTH MUSCLE CONTRACTION IN ARTERIES AND ARTERIOLES OF RAT, GUINEA-PIG DOG AND RABBIT

1. These studies describe the functional effects of modulation of the sarcoplasmic reticulum (SR) Ca²⁺ stores at three levels of the vasculature: (i) large arteries (rat and guinea-pig aorta); (ii) small resistance arteries (rat tail artery, rabbit mesenteric artery, dog mesenteric artery); and (iii) arterioles (guinea-pig submucosal arterioles of the small intestine). 2. All tissues responded to phenylephrine (PE; 10 μmol/L) with a transient contraction in Ca²⁺-free Krebs', reflecting Ca²⁺ release from PE-sensitive Ca²⁺ stores. After pretreatment with cyclopiazonic acid (CPA; 30 μmol/L) or thapsigargin (TSG; 1 μmol/L), putative SR Ca²⁺ pump inhibitors, the PE-induced contraction in a Ca²⁺-free medium was significantly inhibited in arterial tissues at all levels of the vasculature. Similarly, ryanodine (RYA; 30 μmol/L), an agonist that enhances Ca²⁺ release from the SR, also reduced the PE contraction in a Ca²⁺-free solution. 3. CPA or TSG alone in the presence of extracellular Ca²⁺, caused marked and sustained contraction in the rat and guinea-pig aorta and marked but transient or no contraction in the resistance arteries. In the rat and guinea-pig aorta, RYA caused a slowly developing tension. Little increase in basal tension was produced by RYA in resistance arteries and arterioles. 4. The findings show that an agonist-releasable Ca²⁺ pool is present at all levels of the vasculature that is independent of the size of the vessels and suggest that under normal physiological conditions there is an intimate balance between the roles of the plasma membrane and of the SR in the maintenance of vascular contractility. It appears that the role of the SR diminishes as the arteries become smaller, while Ca²⁺ fluxes across the plasma membrane predominates.     

In vitro vasodilator mechanisms of the indole alkaloids rhynchophylline and isorhynchophylline,isolated from the hook of<Emphasis Type="Italic"> Uncaria rhynchophylla</Emphasis> (Miquel)

Rhynchophylline (Rhy) and isorhynchophylline (Isorhy), indole alkaloids from Uncaria hooks, reportedly exert hypotensive and vasodilatory effects, but the mechanism of action is unclear. We therefore investigated the relaxant effects of these two isomeric alkaloids in rat arteries in vitro, in particular in respect of the various functional Ca²⁺ pathways. Both Rhy and Isorhy relaxed aortic rings precontracted with phenylephrine (PE, 1 µM) in a dose-dependent manner (3–300 µM). Removal of endothelium and preincubation with L-NAME (300 µM) slightly inhibited but did not prevent the relaxant response. These results indicate that Rhy and Isorhy act largely in an endothelium-independent manner. Unlike nicardipine, both alkaloids not only inhibited the contraction induced by 60 mM KCl (IC₅₀ 20–30 µM), but also that induced by PE and U46619, albeit to a lesser extent (IC₅₀ 100 and 200 µM, respectively). These results suggest that Rhy and Isorhy may act via multiple Ca²⁺ pathways. In contrast to their inhibitory effects on KCl-induced and receptor-mediated contractions, where both isomers were comparably potent, Rhy was more potent than Isorhy at higher concentrations (>100 µM) in inhibiting both caffeine (25 mM)- and cyclopiazonic acid (CPA, 30 µM)-induced contractions. Similar results observed with caffeine in Ca²⁺-containing medium were also observed in Ca²⁺-free medium. However, 0.1–0.3 µM nicardipine (which completely inhibited KCl-induced contraction) had no significant inhibitory effect on CPA-induced contractions. Taken together, these results indicate discrimination between these two isomers with respect to Ca²⁺-induced Ca²⁺ release and non-L-type Ca²⁺ channel, but not for IP₃-induced Ca²⁺ release and L-type Ca²⁺ channels. Similar relaxant responses to KCl- and caffeine-induced contractions were seen when these two alkaloids were tested on the smaller mesenteric and renal arteries. In conclusion, the vasodilatory effects of Rhy and Isorhy are largely endothelium independent and are mediated by L-type Ca²⁺ channels. At higher concentrations, they also affect other Ca²⁺-handling pathways, although to a lesser extent. While there is no discrimination between the two isomers with respect to the contraction induced by KCl or agonists (PE and U46619), differential effects between Rhy and Isorhy were seen on caffeine- and CPA-induced contractions.     

Participation of both intracellular free Ca2+ and protein kinase C in tonic vasoconstriction induced by prostaglandin F2α

The roles of intracellular free Ca²⁺ and protein kinase C in the tonic contraction induced by prostaglandin were studied. Prostaglandin F_2α induced tonic contraction of rat thoracic aorta in both control and Ca²⁺-free solution. Close correlations were observed between the contractile response of aortic strips and the changes in intracellular free Ca²⁺ concentration in vascular smooth muscle cells assessed with the fluorescent Ca²⁺ indicator fura 2, both in control and Ca²⁺-free solutions. Prostaglandin F_2α also enhanced the production of inositol 1,4,5-triphosphate in vascular smooth muscle cells before the rise of the intracellular free Ca²⁺ concentration. Moreover, 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine, an inhibitor of protein kinase C, inhibited the tonic contractions induced by PGF_2α and 12-O-tetradecanoyl phorbol-13-acetate, a direct activator of protein kinase C, at similar concentrations. These results suggest that both intracellular free Ca²⁺ and protein kinase C participate in prostaglandin F_2α-induced tonic contraction.     

3′,4′-Dihydroxyflavonol reduces vascular contraction through Ca2+ desensitization in permeabilized rat mesenteric artery

3′,4′-Dihydroxyflavonol (DiOHF) exerts endothelium-independent relaxation in rat aortic rings. In this study, we hypothesized that DiOHF reduces vascular contraction through Ca²⁺ desensitization in permeabilized third-order branches of rat mesenteric arteries. The third-order branches of rat mesenteric arteries were permeabilized with β-escin and subjected to tension measurement. Cumulative addition of phenylephrine (0.3–30 μM) produced concentration-dependent vascular contraction of endothelium-intact and endothelium-denuded arterial rings, which were inhibited by pretreatment with DiOHF (10, 30, or 100 μM). In addition, DiOHF dose-dependently decreased vascular contractions induced by 3.0 μM phenylephrine. β-Escin-permeabilized third-order branches of mesenteric arteries were contracted with Ca²⁺, NaF, or guanosine-5′-(γ-thio)triphosphate (GTPγS) 30 min after pretreatment with DiOHF or vehicle. Pretreatment with DiOHF for 30 min inhibited vascular contraction induced by cumulative additions of Ca²⁺ (pCa 9.0–6.0) or NaF (4.0–16.0 mM) in permeabilized arterial rings. Cumulative addition of DiOHF also reduced vascular contraction induced by Ca²⁺-controlled solution of pCa 6.0, 16.0 mM NaF, or 100 μM GTPγS in permeabilized arterial rings. DiOHF inhibited the increase in vascular tension provoked by calyculin A, even though it did not affect vascular tension already produced by calyculin A. DiOHF accelerated the relaxation induced by rapidly lowering Ca²⁺. DiOHF reduced vascular contraction through Ca²⁺ desensitization in permeabilized third-order branches of rat mesenteric arteries. These results suggest that DiOHF may have a therapeutic potential in the treatment of cardiovascular diseases.     

Inhibitory effects of protein kinase inhibitors and cytoskeletal inhibitors on Ca2+-free contraction of rat uterus

In rat uterine smooth muscle, sustained Ca²⁺-free contraction was observed by oxytocin in Ca²⁺-free solution. This Ca²⁺-free contraction was effectively inhibited by protein kinase inhibitors and cytoskeletal inhibitors but myosin-light chain kinase (MLCK) inhibitors were not so effective. Simultaneous addition of a protein kinase inhibitor and a cytoskeletal inhibitor caused synergestic inhibition. These results suggest that the mechanism for Ca²⁺-free contraction involves some protein kinase and cytoskeletal elements rather than MLCK.     

CaMKIIδ and cardiomyocyte Ca2+ signalling new perspectives on splice variant targeting

Control of cardiomyocyte cytosolic Ca²⁺ levels is crucial in determining inotropic status and ischemia/reperfusion stress response. Responsive to fluctuations in cellular Ca²⁺, Ca²⁺/calmodulin‐dependent protein kinase II (CaMKII) is a serine/threonine kinase integral to the processes regulating cardiomyocyte Ca²⁺ channels/transporters. CaMKII is primarily expressed either in the δ_B or δ_C splice variant forms, which may mediate differential influences on cardiomyocyte function and pathological response mechanisms. Increases in myocyte Ca²⁺ levels promote the binding of a Ca²⁺/calmodulin complex to CaMKII, to activate the kinase. Activity is also maintained through a series of post‐translational modifications within a critical region of the regulatory domain of the protein. Recent data indicate that the post‐translational modification status of CaMKIIδ_B/δ_C variants may have an important influence on reperfusion outcomes. This study provided the first evidence that the specific type of CaMKII post‐translational modification has a role in determining target selectivity of downstream Ca²⁺ transporters. The study was also able to demonstrate that the phosphorylated form of CaMKII closely co‐localizes with CaMKIIδ_B in the nuclear/myofilament fraction, contrasting with a co‐enrichment of oxidized CaMKII in the membrane fraction with CaMKIIδ_C. It has also been possible to conclude that a hyper‐phosphorylation of CaMKII (Thr287) in reperfused hearts represents a hyper‐activation of the CaMKIIδ_B, which exerts anti‐arrhythmic actions through an enhanced capacity to selectively increase sarcoplasmic reticulum Ca²⁺ uptake and maintain cytosolic Ca²⁺ levels. This suggests that suppression of global CaMKIIδ may not be an efficacious approach to developing optimal pharmacological interventions for the vulnerable heart.     

Cellular calcium regulatory machinery of vasorelaxation elicited by petasin

1. The present study examined the cytosolic Ca²⁺ regulatory machinery involved in the vasorelaxation produced by petasin, a sesquiterpene isolated from Petasites formosanus. 2. Aortic rings isolated from Sprague‐Dawley rats were exposed to petasin (0.01–100 μmol/L) to elucidate its vascular effects on isometric contraction elicited by vasoconstrictors, as well as the contribution of the endothelium and Ca²⁺ to the responses observed. In addition, L‐type voltage‐dependent Ca²⁺ channel (VDCC) activity and [Ca²⁺]_i were determined in cultured vascular smooth muscle cells (VSMCs) from Sprague‐Dawley rats in the presence of 1–100 μmol/L petasin using whole‐cell patch‐clamp recording and the fluorescent probe fura‐2/AM. The effects of petasin on vascular responses were compared between aortic rings from spontaneously hypertensive rats (SHR) and normotensive Wistar‐Kyoto (WKY) rats. 3. Petasin reduced isometric contraction elicited by KCl or the L‐type Ca²⁺ channel opener BayK 8644 (IC₅₀ 3.0 ± 0.4 and 4.1 ± 1.1 μmol/L, respectively) in aortic rings isolated from Sprague‐Dawley rats, independent of the endothelium. In addition, petasin triggered a rightward shift in the concentration–response curve to KCl while reducing the maximal response by 82%. In Ca²⁺‐depleted and high K⁺‐depolarized aortic rings, 1–100 μmol/L petasin pretreatment attenuated the Ca²⁺‐induced contraction in a concentration‐dependent manner. 4. In cultured VSMCs, whole‐cell patch‐clamp recording revealed that petasin inhibited VDCC activity. Measurement of [Ca²⁺]_i using fura‐2/AM fluorescence indicated that petasin suppressed the KCl‐induced increase in [Ca²⁺]_i. However, receptor binding assays failed to identify any significant interaction between petasin and the dihydropyridine binding sites of the L‐type VDCC. 5. In aortic rings from SHR and WKY rats, petasin inhibited Ca²⁺‐induced contractions in Ca²⁺‐depleted and high K⁺‐depolarized solution with a more pronounced effect in rings from SHR. 6. Together, the results suggest that direct Ca²⁺ antagonism of L‐type VDCC in vascular smooth muscle may account, at least in part, for petasin‐induced vasorelaxation. The more pronounced effect of the sesquiterpene in blood vessels from SHR suggests its possible therapeutic potential in the mangement of hypertension.     

Variable Contribution of TMEM16A to Tone in Murine Arterial Vasculature

TMEM16A is essential for Ca²⁺‐activated Cl^? conductance in vascular smooth muscle. The importance of TMEM16A for agonist‐induced vascular constriction and blood pressure control is, however, under debate. Previous studies suggested that TMEM16A might have a complex cellular function beyond being essential for the Ca²⁺‐activated Cl^? conductance, for example modulation of Ca²⁺ channel expression. Mice with constitutive, smooth muscle‐specific expression of siRNA directed against Tmem16a (transgenic mice, TG) were generated. Isometric constrictions of isolated aorta, mesenteric, femoral and tail arteries from TG mice were compared with wild‐types. Protein expression was analysed by Western blots. Blood pressure and heart rate were studied telemetrically. Significant TMEM16A down‐regulation was seen in aorta and tail arteries, while no changes were detected in mesenteric and femoral arteries. Contractile responses of mesenteric and femoral arteries from TG and wild‐type mice were not different. Aorta from TG mice showed reduced agonist‐induced constriction, while their responses to elevated K⁺ were unchanged. Tail arteries from TG mice also constricted less to adrenergic stimulation than wild‐types. Surprisingly, tail arteries from TG mice constricted less to elevated K⁺ too and were more sensitive to nifedipine‐induced relaxation. Consistently, TMEM16A down‐regulation in tail arteries was associated with reduction in CACNA1C protein (i.e. vascular L‐type Ca²⁺ channel) expression. No differences in blood pressure and heart rate between the groups were seen. This study suggests a complex contribution of TMEM16A in vascular function. We suggest that TMEM16A modulates arterial contractility, at least in part, indirectly via regulation of CACNA1C expression.     

Lead Induces Endothelium‐ and Ca2+‐Independent Contraction in Rat Aortic Rings

Abstract: The contractile effect of lead on rat aortic rings was examined. Lead (0.1–3.1 mM) elicited concentration‐dependent but endothelium‐independent contractions, which were unaffected by prazosin (1 μM). The contractile effects of lead were similar when the aortic rings were bathed either in the absence or presence of external Ca²⁺. Lanthanum (1 mM) but not verapamil (1 μM) inhibited the lead contractions; hence non‐L‐calcium channels are involved in such effect. In addition, lead induced contractions on aortic rings incubated in Ca²⁺‐free EGTA‐containing solution for 70 min., an experimental condition in which intracellular Ca²⁺‐stores are depleted. Finally, the contractile effect of lead was not modified by calphostin C (an inhibitor of protein kinase C). In conclusion, the present results suggest that in rat aorta, the lead‐induced contraction is independent of extra‐ and intracellular calcium stores. In addition, the effect of lead is independent of either catecholamines or protein kinase C. It is likely that in rat aorta, lead enters into the smooth muscle cells through non‐L‐calcium channels, and when acting like calcium on the contractile machinery it produces contraction. The differences observed between our results and those obtained by other authors may indicate that the mechanism of the contractile effect of lead varies among the different blood vessels.     

The toxic effects of Bisphenol A on the mouse spermatocyte GC‐2 cell line: the role of the Ca2+‐calmodulin‐Ca2+/calmodulin‐dependent protein kinase II axis

Bisphenol A (BPA), an endocrine‐disrupting chemical (EDC), is known to induce male reproductive toxicity in rodents. However, its toxic effects on the germ cells are still poorly understood. It has been proposed that Ca²⁺ homeostasis and Ca²⁺ sensors, including calmodulin (CaM) and calmodulin‐dependent protein kinase II (CaMKII), play critical roles in spermatogenesis. Therefore, in the present study, we aimed to investigate whether a perturbation in Ca²⁺‐CaM‐CaMKII signaling was involved in the BPA‐induced injury to mouse spermatocyte GC‐2spd (ts) (GC‐2) cells. Our results showed that BPA (range from 0.2 to 20 μM) induced obvious GC‐2 cell injury, including decreased cell viability, the release of mitochondrial cytochrome c and the activation of caspase‐3. However, these processes could be partially abrogated by pretreatment with a Ca²⁺ chelator (BAPTA/AM), a CaM antagonist (W7) or a CaMKII inhibitor (KN93). These results, taken together, indicate that BPA exposure contributes to male germ cell injury, which may be partially mediated through a perturbation in Ca²⁺/CaM/CaMKII signaling and the mitochondrial apoptotic process. Copyright © 2015 John Wiley & Sons, Ltd.     

Hypertonic saline inhibits airway smooth muscle contraction by inhibiting Ca2+ sensitization

The effects of hypertonic solution on airway smooth muscle (ASM) contraction and the underlying mechanisms are largely unknown. We found that hypertonic saline (HS) inhibited acetylcholine (ACh)‐induced contraction of ASM from the mouse trachea and human bronchi. In single mouse ASM cells (ASMCs), ACh induced an increase in intracellular Ca²⁺ that was further enhanced by 5% NaCl, indicating that the HS‐induced inhibition of ASM contraction was not mediated by a decrease in cytosolic Ca²⁺. The Rho‐associated kinase (ROCK) inhibitor Y‐27632 relaxed ACh‐induced precontraction of mouse tracheal rings. However, such inhibition was not observed after the relaxation induced by 5% NaCl. Moreover, the incubation of mouse tracheal rings with 5% NaCl decreased ACh‐induced phosphorylation of myosin light chain 20 and myosin phosphatase target subunit 1. These data indicate that HS inhibits the contraction of ASM by inhibiting Ca²⁺ sensitization, not by decreasing intracellular Ca²⁺.     

Contraction of the sheep middle cerebral,pulmonary and coronary arteries initiated by release of intracellular calcium

1 The aim of the study was to compare contraction initiated by intracellular Ca²⁺ release in the middle cerebral, coronary and pulmonary arteries of the sheep. With all three arteries from the sheep, incubation in Ca²⁺-free physiological salt solution (PSS) reduced agonist-induced contraction much more than occurred with the rabbit aorta. The intracellular Ca²⁺ store appeared to be of limited capacity, since contraction was transient in Ca²⁺-free conditions with most agonists. 2 In the middle cerebral artery, contraction in Ca²⁺-free conditions was much reduced if a previous contraction had been obtained (for 5-hydroxytryptamine, 5-HT, from 11 + 4 to 1 + 0.5% of control contraction in 2.5 mm Ca²⁺), suggesting that the previous contraction had partly discharged the intracellular Ca²⁺ store. Contraction was less affected in the pulmonary artery and almost unaffected in the coronary artery (for 5-HT, from 15 + 1 to 11 + 1%) by a previous contraction in Ca²⁺-free conditions. 3 Rings prepared from small branches of the pulmonary and coronary arteries were affected by Ca²⁺ deprivation in a similar manner to large diameter pulmonary and coronary artery rings. 4 In Ca²⁺-free PSS, contraction induced by prostaglandin E₂ was almost eliminated (3 + 1% of control contraction in 2.5 mm Ca²⁺), contractions induced by 5-HT and noradrenaline were reduced, and contraction induced by the thromboxane mimetic U46619 was least affected (up to 73 + 8%). 5 Increasing agonist concentration from EC₅₀ to the maximally effective concentration raised the percentage contraction remaining in the middle cerebral artery (for noradrenaline from 7 + 2% to 12 + 3%) but not in the pulmonary artery (for noradrenaline from 22 + 2% to 24 + 6%). 6 The present study has revealed notable differences, in coupling to intracellular Ca²⁺ release between the three vascular territories studied.     

Rho kinase and protein kinase C involvement in vascular smooth muscle myofilament calcium sensitization in arteries from diabetic rats

Background and purpose:

Diabetes mellitus (DM) causes multiple dysfunctions including circulatory disorders such as cardiomyopathy, angiopathy, atherosclerosis and arterial hypertension. Rho kinase (ROCK) and protein kinase C (PKC) regulate vascular smooth muscle (VSM) Ca²⁺ sensitivity, thus enhancing VSM contraction, and up-regulation of both enzymes in DM is well known. We postulated that in DM, Ca²⁺ sensitization occurs in diabetic arteries due to increased ROCK and/or PKC activity.

Experimental approach:

Rats were rendered hyperglycaemic by i.p. injection of streptozotocin. Age-matched control tissues were used for comparison. Contractile responses to phenylephrine (Phe) and different Ca²⁺ concentrations were recorded, respectively, from intact and chemically permeabilized vascular rings from aorta, tail and mesenteric arteries.

Key results:

Diabetic tail and mesenteric arteries demonstrated markedly enhanced sensitivity to Phe while these changes were not observed in aorta. The ROCK inhibitor HA1077, but not the PKC inhibitor chelerythrine, caused significant reduction in sensitivity to agonist in diabetic vessels. Similar changes were observed for myofilament Ca²⁺ sensitivity, which was again enhanced in DM in tail and mesenteric arteries, but not in aorta, and could be reduced by both the ROCK and PKC blockers.

Conclusions and implications:

We conclude that in DM enhanced myofilament Ca²⁺ sensitivity is mainly manifested in muscular-type blood vessels and thus likely to contribute to the development of hypertension. Both PKC and, in particular, ROCK are involved in this phenomenon. This highlights their potential usefulness as drug targets in the pharmacological management of DM-associated vascular dysfunction.     

Involvement of different Ca2+ pools during the canine bronchial sustained contraction in Ca2+-free medium: lack of effect of PKC inhibition

We evaluated the role of protein kinase C (PKC) in the sustained bronchial contraction (SBC) induced by carbachol (Cch) or histamine in a Ca²⁺-free medium and the possibility that each agonist uses a different Ca²⁺ store for this response. We studied third-order bronchi and airway smooth muscle (ASM) from first-order bronchi dissected free of cartilage and epithelium. Bronchial and ASM responsiveness to Cch or histamine were evaluated in Krebs solution (2.5 mM Ca²⁺) and in Ca²⁺-free medium. Cch and histamine induced an SBC in bronchial tissues in Ca²⁺-free medium. In ASM each agonist produced a transient contraction, but the response to histamine was much smaller. Cch induced a concentration-dependent accumulation of inositol phosphates (IPs) in both bronchi and ASM; however, histamine did not induce significant accumulation of IPs. Repeated exposure to histamine in bronchial rings abolished contractile responses in Ca²⁺-free media, but Cch added afterwards still produced a sustained contraction. This response was blocked when bronchial tissues were preincubated with 10 μM cyclopiazonic acid (CPA). Brief incubation of these preparations with a high EGTA concentration (1 mM) abolished the histamine-induced SBC. The SBC induced by Cch or histamine in Ca²⁺-free medium was not affected by the preincubation of the tissues with calphostin C, chelerythrine or staurosporine. We concluded that Cch mobilizes Ca²⁺ from two different sources during the SBC in Ca²⁺-free medium: from a CPA-sensitive one from sarcoplasmic reticulum (SR) and from a putative extracellular membrane Ca²⁺ pool sensitive to 1 mM EGTA, and neither process involved PKC activation. Histamine appeared to utilize the extracellular membrane pool only. Received: 12 March 1998 / Accepted: 2 September 1998     

Alzheimer's disease therapeutic candidate SAK3 is an enhancer of T-type calcium channels

Low-threshold Ca²⁺ spikes are mediated by T-type Ca²⁺ channels, which have electrophysiological properties of fast inactivation and slow deactivation kinetics. A low membrane potential of approximately ?60 mV is sufficient to trigger channel opening. We recently introduced a novel T-type Ca²⁺ channel enhancer that improves cognition and inhibits amyloid beta aggregation in an Alzheimer's disease (AD) mouse model. The enhancer stimulates ACh release, Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) activity, and neurogenesis in the hippocampus. Then, we discuss how T-type Ca²⁺ channel enhancer improves cognition and impaired neurogenesis and how CaMKII signaling in neurodegenerative diseases reduces amyloid beta aggregation. We provide a perspective of the potential AD therapies to target CaMKII signaling. In this context, we overview our attempts leading to the development of a T-type Ca²⁺ channel enhancer as cognitive enhancer, the action of which has been associated with CaMKII and presumably proteasome activity.     

Oxidative activation of the Ca2 +/calmodulin-dependent protein kinase II (CaMKII) regulates vascular smooth muscle migration and apoptosis

Activation of the Ca^2 +/calmodulin-dependent protein kinase II (CaMKII) and reactive oxygen species (ROS) promote neointimal hyperplasia after vascular injury. CaMKII can be directly activated by ROS through oxidation. In this study, we determined whether abolishing the oxidative activation site of CaMKII alters vascular smooth muscle cell (VCMC) proliferation, migration and apoptosis in vitro and neointimal formation in vivo. VSMC isolated from a knock-in mouse with oxidation-resistant CaMKIIδ (CaMKII M2V) displayed similar proliferation but decreased migration and apoptosis. Surprisingly, ROS production and expression of the NADPH oxidase subunits p47 and p22 were decreased in M2V VSMC, whereas superoxide dismutase 2 protein expression was upregulated. In vivo, after carotid artery ligation, no differences in neointimal size or remodeling were observed. In contrast to VSMC, CaMKII expression and autonomous activity were significantly higher in M2V compared to WT carotid arteries, suggesting that an autoregulatory mechanism determines CaMKII activity in vivo. Our findings demonstrate that preventing oxidative activation of CaMKII decreases migration and apoptosis in vitro and suggest that CaMKII regulates ROS production. Our study presents novel evidence that CaMKII expression in vivo is regulated by a negative feedback loop following oxidative activation.