mM Of KCl Research Articles

Vasoconstrictive Effect of Angiotensin IV in Isolated Rat Basilar Artery Independent of AT1 and AT2 Receptors

The effect of angiotensin IV (AngIV) was studied in freshly isolated rat basilar arteries (BAs) perfused at a constant rate. AngIV had no effect on basal BA perfusion pressure, but induced a marked concentration-dependent contraction in vessels precontracted by a 50-mM KCl solution (EC₅₀ = 44.5 ± 16 nM). This contraction was unaffected by the angiotensin AT₁ receptor antagonist candesartan or the angiotensin AT₂ receptor blocker PD123319, but was markedly inhibited by two different specific AT₄ receptor antagonists, Nle¹-Leu³¥(CH₂-NH₂)^3–4-AngIV and divalinal-AngIV. Removal of the endothelium abolished the contractile response to AngIV, and pretreatment of endothelium-intact arteries with the endothelin ET_A/ET_B receptors inhibitor PD142893 blocked the AngIV-induced contraction to the same extent. In BA pretreated with endothelin-1 (ET-1; 0.01 µM), AngIV-induced a concentration-dependent contraction, shifted to the left, compared with that observed with KCl precontraction, unaffected by candesartan but completely abolished by Nle¹-Leu³¥(CH₂-NH₂)^3–4-AngIV. The contractile effect was not affected by endothelium removal in the presence of exogenous ET-1, in contrast to KCl pretreated BA, suggesting that endothelium was mandatory to unmask the effect of AngIV as a source of endogenous ET-1 release. Taken together, these results indicate that low (nanomolar) concentrations of AngIV exert a constrictive effect mediated by its specific binding site AT₄ in the rat BA, and that this vasoactive effect is indirect and involves endogenous endothelin(s).

Reversed micellar extraction of an extracellular protease from Nocardiopsis sp. fermentation broth

Extraction of an extracellular alkaline protease from Nocardiopsis sp. fermentation broth using reversed micelles of sodium di(2-ethylhexyl) sulfosuccinate (AOT) in isooctane was performed with equal phase volume ratio. This work describes the effects of pH, ionic strength and surfactant concentration on the enzyme transfer process from the aqueous to the organic phase by direct contact (5 min) between the two phases. The best conditions for extraction (38.4% of protein content with about 83.5% of activity) were obtained using 200 mM AOT, at pH 4.0 with 50 mM KCl. For back extraction, sodium carbonate buffer, at pH 5.0 with 100 mM of KCl allowed for the best conditions (47.2% of protein content with about 14.0% of activity). The low protease activity yield of 11.69% obtained for the total process, extraction and back-extraction, suggests enzyme denaturation or its allocation near the AOT hydrophobic tail.

Avidin self‐associates with boric acid gel suspensions: preparation of an affinity boron carrier that might be developed for boron‐neutron capture therapy

AbstractIt has been shown in preliminary studies that avidin self‐associates with suspended arylboronic acid gel polymer particles, and avidin‐coated gel particles in the micron and submicron size range are of interest to boron‐neutron capture therapy (BNCT) applications because they carry sufficiently large amounts of boron‐10 to effect a meaningful tissue dosing through BNCT. Avidin‐coated gel particles precipitate within 46 min of mixing when the avidin/colloid ratio is about 0.34 g avidin/g colloid. This is a minimum time if gel and avidin concentrations are in the low microgram/milliliter range, and further increases in the relative amount of avidin increase the time to precipitation. At shorter exposure times rapid agglutination‐like reactions were observed with multivalent biotinylated bovine albumin, suggesting that two stage pre‐targeting of specific tissues should be possible with biotinylated antitumor antibodies. The yield of micron sized and smaller gel particles is enhanced by a combination of manual grinding in an agate mortar followed by sonification. Sonification alone produces an uneven distribution, with fewer particles sedimenting at 15 000 rpm than above or below. Preparation of avidin‐coated boronic acid particles is a matter of combining a phosphate buffered solution of avidin (120 mM in NaCl, 2.7 mM in KCl and 10 mM in phosphate, pH 7.4) and an aqueous suspension of gel particles in the mass ratio described above. Copyright © 2005 John Wiley & Sons, Ltd.

Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator Channel by β-Adrenergic Agonists and Vasoactive Intestinal Peptide in Rat Smooth Muscle Cells and Its Role in Vasorelaxation

Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator Channel by β-Adrenergic Agonists and Vasoactive Intestinal Peptide in Rat Smooth Muscle Cells and Its Role in Vasorelaxation

Caffeic acid phenethyl ester blocks apoptosis induced by low potassium in cerebellar granule cells

Primary cultures of cerebellar granule neurons (CGNs) were prepared from 8-day-old Wistar rats, and maintained in an appropriate medium containing a high (25mM) concentration of KCl. To induce apoptosis, culture medium was replaced with serum-free medium (containing 5mM KCl) 8 days after plating. Apoptosis was measured by the terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick end-labeling (TUNEL) method, and by flow cytometry. Since there is evidence that an increased formation of reactive oxygen species (ROS) is involved in the apoptosis induced by low K+ (5mM) concentrations, the potential anti-apoptotic effect of caffeic acid phenethyl ester (CAPE), a potent flavonoid antioxidant, was tested in this experimental model. It was found that CAPE (10μg/ml) promoted cell survival and was capable of blocking the apoptotic process as assayed by both TUNEL and flow cytometric methods. The same concentration of CAPE prevented the formation of ROS induced by low K+. Since there is evidence that low K+-induced apoptosis in CGNs is associated with a drop in intracellular Ca2+ concentration ([Ca2+]i), activation of the cell death effector proteases caspase-3 and caspase-9, and of the transcription factor nuclear factor kappa B (NF-κB), the interference of CAPE with these purported mediators of apoptosis was also evaluated. It was found that CAPE did not interfere with the marked decrease in [Ca2+]i induced by low K+, whereas it completely blocked caspase-3, caspase-9, and NF-κB activation. It is concluded that CAPE could exert its anti-apoptotic effect in CGNs by blocking ROS formation and by inhibiting caspase activity.

Mechanisms of cell death by deprivation of depolarizing conditions during cerebellar granule neurons maturation

Cerebellar granule cells (CGC) cultured under 5mM KCl (K5) undergo apoptosis after 5 days in vitro (DIV). CGC death is reduced by chronic treatment with 25 mM KCl (K25) or NMDA. Also, when CGC cultured for 6–8 DIV in K25 are transferred to a K5 medium, cells die apoptotically. Moreover, Bcl-2 and Bcl-xL protect neurons from apoptosis, while Bax and Bcl-xS may act as proapototic proteins. It is suggested that these members of the Bcl-2 family may be involved in the cytochrome- c (cyt- c) release to the cytosol. Cytochrome- c is able to form a complex with other proteins to activate a cascade of proteases. In this work, we found that Bcl-2 levels in K5 cells did not show any change during 2–7 days in vitro (DIV); but cells grown with NMDA and K25 displayed an increase (55% approximately) of Bcl-2 from 4 DIV, as compared to control. Under these conditions, Bax levels showed a tendency to decrease with age under control cells and NMDA/K25 induced a reduction of approximately 10% in Bax levels from 4 DIV. On the other hand, in cells maintained in K25 during 7 DIV and then switched to a K5 medium, the levels of Bax showed a consistent decrease (30% after 8 h). Under these conditions, the Bcl-2 levels did not show any significant change after 24 h. Cytochrome- c levels were unaffected under K5, NMDA and K25 and only a marginal increase of cytochrome- c in the cytosol was detected at 6 h after switching. We also found that caspase-9 was only activated under K25-deprivation meanwhile caspase-3 was involved in both protocols. These results suggest that the Bcl-2 family members, caspases activation and cytochrome- c release are involved in CGC death induced by K5 and their participation in this process could be different depending on neuronal maturation in culture.

Endothelium-dependent relaxation to poly-L-arginine in canine coronary arteries.

The endothelium-dependent relaxation elicited by poly-L-arginine was investigated in canine coronary arteries. Poly-L-arginine (10(-11) to 10(-7) M) induced concentration-dependent endothelium-dependent relaxation in segments of canine coronary arteries incubated with indomethacin. In the presence of indomethacin, arteries contracted with prostaglandin F2alpha and exposed also to ouabain, oxyhemoglobin, or N(omega)-nitro-L-arginine, as well as those contracted with 20 mM of KCl, presented reduced endothelium-dependent relaxation to poly-L-arginine. The combination of N(omega)-nitro-L-arginine with indomethacin produced further reduction of endothelium-dependent relaxation in arteries contracted with prostaglandin F2alpha as well as in arteries contracted with 20 mM of KCl. However, only quinacrine or calmidazolium, both in presence of N(omega)-nitro-L-arginine plus indomethacin, abolished the endothelium-dependent relaxation elicited by poly-L-arginine. The relaxation to poly-L-arginine was not significantly affected by glybenclamide. The present study demonstrates that in addition to the prostacyclin and nitric oxide-dependent mechanisms, poly-L-arginine also elicits endothelium-dependent relaxation in canine coronary arteries, stimulating the release of endothelial relaxing factor(s) produced by the phospholipase A2 pathway, but independent of the cyclooxygenase pathway, which may cause hyperpolarization of the vascular smooth muscle.

Monitoring of exocytosis and endocytosis of insulin secretory granules in the pancreatic β-cell line MIN6 using pH-sensitive green fluorescent protein (pHluorin) and confocal laser microscopy

The dynamics of exocytosis/endocytosis of insulin secretory granules in pancreatic β-cells remains to be clarified. In the present study, we visualized and analysed the motion of insulin secretory granules in MIN6 cells using pH-sensitive green fluorescent protein (pHluorin) fused to either insulin or the vesicle membrane protein, phogrin. In order to monitor insulin exocytosis, pHluorin, which is brightly fluorescent at approximately pH7.4, but not at approximately pH5.0, was attached to the C-terminus of insulin. To monitor the motion of insulin secretory granules throughout exocytosis/endocytosis, pHluorin was inserted between the third and fourth amino acids after the identified signal-peptide cleavage site of rat phogrin cDNA. Using this method of cDNA construction, pHluorin was located in the vesicle lumen, which may enable discrimination of the unfused acidic secretory granules from the fused neutralized ones. In MIN6 cells expressing insulin—pHluorin, time-lapse confocal laser scanning microscopy (5 or 10s intervals) revealed the appearance of fluorescent spots by depolarization after stimulation with 50mM KCl and 22mM glucose. The number of these spots in the image at the indicated times was counted and found to be consistent with the results of insulin release measured by RIA during the time course. In MIN6 cells expressing phogrin—pHluorin, data showed that fluorescent spots appeared following high KCl stimulation and remained stationary for a while, moved on the plasma membrane and then disappeared. Thus we demonstrate the visualized motion of insulin granule exocytosis/endocytosis using the pH-sensitive marker, pHluorin.

Characterisation of angiotensin II receptors in isolated human subcutaneous resistance arteries.

Subcutaneous arteries have been used as a model for resistance arteries, which are potentially involved in enhanced blood pressure (BP) regulation in man. Angiotensin II (Ang II) is an important regulator of tone, acting via type 1 (AT1-) and type 2 (AT2-) receptor subtypes. The aim of this study was to characterise the Ang II receptors in isolated human subcutaneous arteries, using pharmacological and molecular methods. Subcutaneous arteries were obtained from patients undergoing elective gut surgery and were carefully dissected from the abdominal wall. Cylindrical segments were mounted on two L-shaped metal prongs, one of which was connected to a force-displacement transducer for continuous recording of isometric tension. Concentration-response curves to Ang II were constructed in the presence and absence of various selective AT1-receptor antagonists, candesartan, EXP3174, irbesartan and losartan, and the AT2-receptor antagonist, PD 123319. Responses to Ang II were measured as increases in force (mN) and expressed as a percentage of the response to 60 mM of KCl. Ang II caused a concentration-dependent contraction (pEC50=9.45±0.48, Emax=120±13%). Candesartan and EXP3174 caused concentration-dependent depression of the Emax of Ang II without any major shift of pEC50. Losartan and irbesartan caused a significant, dose-dependent rightward shift of the Ang II contraction-response curve in human subcutaneous arteries. The results show that contractile responses of human subcutaneous arteries are mediated via the AT1-receptor. The AT1-receptor antagonists, candesartan and EXP3174, acted in an insurmountable manner, while losartan and irbesartan were surmountable AT1-receptor antagonists. The AT2-receptor antagonist, PD 123319, (10, 100 nM) had no effect on Ang II-induced contraction. This is supported by the positive identification of mRNA for the human AT 1-receptor by RT-PCR.

Systemic administration of mexiletine for attenuation of cerebral vasospasm following experimental subarachnoid haemorrhage.

Mexiletine is a class Ib drug that is widely used to treat ventricular arrhythmias. This compound is mainly known as a sodium channel blocker, but studies have demonstrated that it can also activate ATP-sensitive K+ channels and block Ca2+ channels. Recent in vitro data from experiments on liposomes indicate that mexiletine is also a potent antioxidant. The unique activity profile of this drug raised the possibility that it might be of benefit in limiting cerebral vasospasm. Our first series of experiments assessed the effects of mexiletine on transclivally exposed rabbit basilar arteries. The arteries were treated with 50-mM KCl, 20-nM endothelin-1 (ET-1), or 100-microM lysophosphatidic acid (LPA) in the presence or absence of 400-mM mexiletine. Vasoconstriction caused by KCl, ET-1, and LPA was inhibited by mexiletine. In a second series of experiments, subarachnoid haemorrhage (SAH) was induced in rabbits by injecting 3-ml of autologous arterial blood into the cisterna magna. Forty-eight hours after SAH induction, transclivally exposed basilar arteries exhibited a spastic constriction that was partially reversed by topical application of 400-microM mexiletine. In a third set of experiments, mexiletine was administered orally at dosages of 80-, 20, and 5-mg/kg/day t.i.d., beginning 3 hours before SAH to study the prevention of vasospasm. In a separate group of animals, 80- and 20-mg/kg/day t.i.d. of mexiletine was administered 21 hours post-SAH induction, to study the reversal of vasoconstriction. Microscopic analysis of vessels from controls (no SAH), SAH-only, and SAH + mexiletine groups indicated there was 71.43% vascular constriction in the SAH-only group compared with controls. Considerable vasorelaxation was seen in the prevention study, in which average arterial cross-sectional areas were reduced by only 17.86% and 39.29% in the mexiletine 80- and 20-mg/kg/day groups, respectively, compared with controls (p < 0.001). Compared with controls, average arterial cross-sectional areas were reduced by 53.58% and 64.29% in the mexiletine 80- and 20-mg/kg/day reversal groups, respectively. Our findings indicate that mexiletine induces potent relaxation in cerebrovascular arteries contracted with various agents, and that it prevents and partially reverses SAH-induced vasoconstriction.

A secretory mechanism of renal kallikrein by a high potassium ion; a possible involvement of ATP-sensitive potassium channel

A relatively rapid excretion of urinary kallikrein into urine was observed by an intravenous infusion of high potassium in anesthetized rats. Superfusion of sliced cortex isolated from rat kidney with an isotonic solution containing more than 20 mM of KCl significantly stimulated the release of kallikrein. The latter in vitro result supported another mechanism for the release of renal kallikrein from kidney other than biosynthesis of kallikrein by aldosterone released from adrenal cortex after loading of high potassium and the mechanism was elucidated. ATP-sensitive potassium channel blockers, glibenclamide, 4-morpholinecarboximidine-N-1-adamantyl-N′-cyclohexylhydrochloride (U37883A), and barium chloride, which inhibit an efflux of intracellular potassium to block the channel, showed a significant increase of the kallikrein release from the slice of kidney cortex. Cytochalasin B, which inhibits a polymerization of actin, also showed a stimulation of the release. Enhanced release of kallikrein by a high potassium or ATP-sensitive potassium channel blocker was reduced by the absence of calcium ion and the presence of voltage-dependent calcium channel blocker in the superfused solution. These results indicate the ATP-sensitive potassium channel which couples to voltage-dependent calcium channel and cytoskeletal protein could be involved in a rapid secretory mechanism of renal kallikrein by high potassium.

Application of Cryo-High Resolution (Hr) Sem Technique for Imaging Platelet Morphology

Abstract Platelets play a major role in hemostasis due to their ability to adhere and aggregate at sites of vessel wall injury. Damage to the inner surface of arteries includes ruptures or tears at the site of atherosclerotic plaques or injury resulting from interventional procedures. Uncontrolled platelet recruitment and aggregation at these sites can lead to complete occlusion of the vessel. Thus the understanding of platelet function and structure has become an important goal in preventing vascular disease. We employed cryo-HRSEM techniques to gain more insight into the surface morphology of both quiescent and thrombin stimulated platelets. Platelet rich plasma (PRP) was obtained by mixing whole blood (21 ml) drawn from juvenile pigs with 3 ml of acid citrate dextrose and centrifugation at 300g for 10 min. To obtain washed platelets the PRP was washed three times with a citrate wash buffer consisting of 36mM citric acid, 5mM glucose, 5mM KCl, 2 mM CaCl2, 1 mM MgCl2, 90 mM NaCl, pH 6.5.

Mechanism by which GABA, through its GABA A receptor, modulates glutamate release from rat cortical neurons in culture

In cortical neurons, the GABA A agonist, muscimol, increases: (a) basal glutamate release (with a EC50 of 99±7 mM); (b) intracellular calcium and (c) membrane potential, all of these in a dose-dependent manner. These muscimol effects were specific since they were reversed by bicuculline, a GABA A antagonist. When the action of muscimol was measured at different KCl concentrations, an increase or decrease of the glutamate secretion was observed, depending on the KCl concentration in the medium. At low KCI concentration (5.6 mM of KCl), it depolarized, at 20 mM of KCl it had no effect, but at higher KCl concentrations (30–100 mM of KCl), it produced a hyperpolarization in these cells. The mechanism by which the GABA-Cl −-channel permits Cl − fluxes, inward or outward, depending on the membrane potential.

The Determination of Halides and Pseudohalides by the Vanadium Haloperoxidase Based Biosensor

A biosensor containing vanadium haloperoxidase (VHP) immobilized on a graphite electrode responded to the halides and hydrogen peroxide at electrode potentials from -0.1 to 0.25 V vs SCE. An apparent Michaelis-Menten constant (Km) of the biosensor was 12–14 μM for hydrogen peroxide at 0.1–0.2 mM of KCl and 0.1 mM of sodium vanadate. Km decreased up to 2.3 μM in a vanadate free solution. At the same conditions and at a fixed concentration (0.1 mM) of hydrogen peroxide Km for chloride changed from 0.6 to 2.5 mM. Km for bromide was 9–16 μM and for iodide it was 5.5 μM. The biosensor response was largest at pH 5.1–5.3 and at pH 6.1 in presence of chloride and iodide, respectively. KNO3 in a competitive manner inhibited the biosensor response; Ki was 0.6 and 0.27 mM, for bromide and chloride. Ki for thiocyanate and for the same halides was 0.25 and 0.08 μM. 10.1 μM of cyanide inhibited 50 % of the biosensor response at 5 mM of chloride. Sodium azide irreversibly inhibited the biosensor response at 1–2 nM and in the presence of 10 μM of bromide or 5 mM of chloride.

Electrocatalysis by vanadium haloperoxidase

Abstract A steady-state potential of an enzyme electrode containing immobilized vanadium haloperoxidase (VHP) on graphite reached a value of 0.46–0.60 V versus SCE in a solution containing hydrogen peroxide, vanadate and halide at pH 6.0. The electrocatalytic current of chloride oxidation was practically constant from −0.1 to 0.25 V versus SCE. The current determined at 0 V versus SCE depended on the hydrogen peroxide, halide and vanadate concentration. An apparent Michaelis–Menten constant ( K m ) of the enzyme electrode was 12–14 μM for hydrogen peroxide at 0.1–0.2 mM of KCl and 0.1 mM of vanadate. K m decreased to 2.3 μM in vanadate free solution. Under the same conditions and at a fixed concentration (0.1 mM) of hydrogen peroxide, K m for chloride changed from 0.6 to 2.5 mM; K m for bromide was 9–16 μM and for iodide K m was 5.5 μM. Chloride oxidation was activated, bromide and iodide inhibited by extra addition of 0.1 mM of vanadate. The enzyme electrode current depended on the solution pH. When iodide was oxidized the maximal current was at pH 6.1; the current of chloride oxidation was largest at pH 5.1–5.3. KNO 3 inhibited the electrode current in a competitive manner when bromide or chloride was oxidized. K i was 0.6 and 0.27 mM, for the respective ions. K i for thiocyanate was 0.25 and 0.08 μM, respectively. The results achieved conclude that the electron exchange rate between the electrode and VHP activated by hydrogen peroxide was slow. The electrode potential was the result of an electrochemical hypohalous acid reduction. Parallel hypohalous acid reduction by hydrogen peroxide lowers the electrode potential in comparison to the potential of the hypohalous acid/halide redox pair.

Effect of spermine and alpha-difluoromethylornithine on KCl- and CaCl2-induced contraction in rat uterine smooth muscle.

1. The effect of a polyamine, spermine (0.3-30 mM), and an ornithine decarboxylase inhibitor, alpha-difluoromethylornithine (DFMO; 1-10 mM), given alone and in combination were studied on KCl (33, 60 or 90 mM)-induced tonic contraction and on the cumulative concentration-response curves elicited by CaCl2 (30 microM to 10 mM) in a depolarizing (with 33, 60 or 90 mM of KCl) calcium-free medium in isolated rat uterus. 2. Spermine elicited a concentration-dependent relaxation on KCl 33 mM (IC50 = 2.18 +/- 0.37 mM, n = 6), 60 mM (IC50 = 7.80 +/- 0.79 mM, n = 11) and 90 mM KCl (IC50 = 29.55 +/- 4.08 mM, n = 7) induced tonic contraction. The IC50 values were significantly different (P < 0.01). 3. DFMO relaxed the tonic contraction induced by 33 mM of KCl (Emax = 80.70 +/- 13.01%) but only relaxed the contractions induced by 60 and 90 mM of KCl to 23.60 +/- 4.60% and 16.90 +/- 4.10%, respectively. DFMO (10 mM) did not modify the concentration-dependent relaxation elicited by spermine on KCl (60 mM)-induced tonic contraction. 4. KCl (33, 60 or 90 mM) did not produce contraction in a calcium-free medium, but enabled CaCl2 (30 microM to 10 mM) to induce cumulative concentration-dependent contraction of the rat uterus. The EC50 values for CaCl2 were: 0.74 +/- 0.08 (n = 12), 0.34 +/- 0.03 (n = 14) and 0.48 +/- 0.02 (n = 12) mM in medium with 33, 60 or 90 mM of KCl, respectively. 5. Spermine (1 mM) and DFMO (1 mM) did not modify the concentration-response curves induced by CaCl2 in medium with 33 mM of KCl. Higher concentrations of spermine (3 mM) and DFMO (10 mM) strongly reduced the contractile effect of CaCl2 to 35.69 +/- 3.96% (n = 6) and 40.14 +/- 10.74% (n = 6). This inhibitory effect of spermine and DFMO was prevented by increasing KCl concentration in the medium to 60 or 90 mM. Thus, the Emax of CaCl2 in the presence of spermine (3 mM) was 66.26 +/- 6.96% and 89.02 +/- 2.89% in a 60 and 90 mM KCl medium, respectively. In the presence of DFMO (10 mM) the Emax of CaCl2 reached 100% when KCl was increased. 6. Spermine (1 mM) plus DFMO (1 mM) and spermine (1 mM) plus DFMO (10 mM) produced a synergic inhibitory effect of CaCl2-induced contraction in medium with 33 mM of KCl. Spermine (3 mM) plus DFMO (10 mM) produced a total inhibition of CaCl2-induced contraction. 7. The inhibitory effect of spermine (1 mM) plus DFMO (10 mM) was also prevented by increasing KCl concentration in the medium to 60 or 90 mM. The Emax for CaCl2 (10.9 +/- 5.5%) in the presence of KCl 33 mM increased up to 93.2 +/- 2.1% and 96.3 +/- 1.2% when the KCl concentration in the medium was enhanced to 60 or 90 mM. 8. Our results suggest that a calcium inhibitory effect of spermine and DFMO in isolated rat uterus could be produced, since this was prevented by depolarization, as a result of the increase of KCl concentration in the medium.

Effect of Korean red ginseng on the rabbit corpus cavernosal smooth muscle.

The extracts of Korean red ginseng (EKG) is a complex mixture containing ginsenosides, polysaccharides, and several other products. Animal experiments have shown that the intravenous administration of extract of ginseng reduces blood pressure. Recently, it has been reported that ginseng has a relaxing effect on vascular smooth muscle and that the relaxation is associated with nitric oxide (NO) released from the vascular endothelium. The present study was undertaken to investigate the effects of EKG on isolated rabbit corpus cavernosal smooth muscle for evaluation of the possibility of developing EKG as an pharmacoerecting agent. Strips of rabbit corpus cavernosum were mounted in organ chambers to measure isometric tension. On the precontracted muscle strip with phenylephrine (PHE; 5 x 10(-6) M), EKG began to exert a relaxing effect at the concentration of 1 mg/ml and the maximal relaxation effect was reached at 40 mg/ml in a dose-dependent manner. EKG was inhibited significantly by endothelial disruption and by pretreatment with methylene blue, pyrogallol, L-NNA or atropine. EKG partially inhibited the PHE (5 x 10(-6) M) induced contraction up to 45.67% of the control in a dose-dependent fashion. EKG decreased basal tension as well as inhibited the contraction induced by addition of CaCl2 (10(-3) M) dose-dependently in muscle strips at basal equilibrated state in Ca2+ free, high K+ depolarizing solution. EKG also inhibited the contraction induced by depolarization with 20, 40 and 60 mM of KCl. However, this inhibitory effect did not occur with high concentrations of KCl (80 and 120 mM). EKG has a relaxing effect on the rabbit corpus cavernosal tissue in a dose dependent manner. The relaxation action of EKG is mediated by multiple action mechanisms that include increasing the release of NO from the corporal sinusoids, increasing intracellular calcium sequestration, and a hyperpolarizing action.

Isolated human chorionic vascular reactivity: Technical considerations for fresh preparations

1. 1. Sixty chorionic vascular rings from normal term placentas were immersed in an organ bath for isometric tension recording to study (A) the contractile response to 120 mM of potassium chloride (KCI) after adjustment and equilibration to 1–5 g of passive tension; and (B) the concentration-response curve to KCI after adjustment and equilibration to the optimal passive tension. 2. 2. Adjustment to 4 g of passive tension elicited the maximal ( P<0.007) and the latest ( P<0.006) KCl-induced contraction among arterial rings. Venous rings showed the greatest contraction when adjusted to 3 g, but the differences were not significant except when compared to 1 g of passive tension ( P<0.03). 3. 3. The EC 50 for chorionic arteries and veins was 14.2 and 25.7 mM, respectively ( P<0.003). The maximal contraction was already obtained with 40 mM of KCl. 4. 4. Our results suggest that (A) the optimal passive tension for fresh human chorionic arteries is 4 g; (B) chorionic venous reactivity is less influenced by the initial tension; and (C) the optimal concentration of KCl to be used as a contracting agent of these tissues is 40 mM.

Nitric Oxide Inhibition Simulates the Enhancement of α1Agonist-Induced Vasoconstriction in Diabetes

We have previously reported that endothelium-dependent, nitric oxide (NO)-mediated vasorelaxation is impaired in diabetic mesenteric arteries. We hypothesized that vasoconstrictor responses should therefore be enhanced. The purpose of this study was to determine whether diabetic mesenteric arteries exhibit increased vasoconstrictor responses, and to investigate if these changes are receptor and/or NO mediated. Thirty age-matched male Sprague–Dawley rats were divided into control (C) and diabetic (D, streptozotocin: 60 mg/kg) groups and studied after 4 weeks. Terminal branches of ileal mesenteric arteries (300 ± 9 μm) were isolated, pressurized, and superfused with modified Krebs solution. Changes in vessel internal diameter were measured and dose–response curves (DRC) for each vasoactive agent were determined. Each vessel was initially constricted with 40 mMof KCl to determine maximal vasoconstriction. Phenylephrine (Phe, 10−8–10−4M) and UK14304 (10−9–10−5M) were used to determine α1- and α2-receptor responses, respectively. Similar studies were performed in the presence ofNω-nitro-l-arginine methyl ester (l-NAME, 10−4M), a competitive inhibitor of NO synthase. Maximal response (Max), area under the curve (AUC), and vessel sensitivity (ED50) for each DRC were calculated. Comparisons among groups were made using analysis of variance and Student'sttest with Bonferroni correction. There were no differences in vasoconstrictor responses induced by KCl (C: 82 ± 2% vs D: 80 ± 1%). α1-vasoconstrictor responses to Phe were enhanced in diabetes with significantly higher Max (96 ± 2% vs 83 ± 3%), and AUC (1.92 ± 0.09 vs 1.56 ± 0.08), but no difference in ED50. The addition ofl-NAME enhanced only Phe-induced vasoconstrictor response significantly in control rats. Thus, differences in Phe-induced vasoconstrictor responses between C and D were abolished in the presence ofl-NAME. α2-vasodilator responses induced by UK14304 were similar between C and D and unaffected byl-NAME. α1-, but not α2-, vasoconstrictor responses are enhanced in streptozotocin-induced diabetic rats. These enhanced responses can be duplicated by treatment of control vessels withl-NAME.

Characteristics of Muskellunge Spermatozoa II: Effects of Ions and Osmolality on Sperm Motility

We investigated the effects of potassium, sodium, glucose, and calcium concentrations, alone or in combinations, on sperm motility in muskellunge Esox masquinongy. Sperm motility was evaluated by the duration of sperm movement and the initial percentage of motile sperm. The osmolality of diluents rather than the specific ions or nonelectrolyte played a major role in the regulation of sperm motility in muskellunge. Sperm were fully activated (>80%) when the concentration was lower than 50 mM of KCl and NaCl, or 100 mM glucose (all in 30 mM tris–HCl at pH 8.0). A small percent of spermatozoa could be activated at 150 mM KCl and NaCl, or 300 mM glucose, which were hypertonic to the seminal plasma. The duration of sperm movement was up to 6–7 min at 12°C in a solution of 100 mM glucose or 50 mM NaCl. Spermatozoa had a prolonged duration of movement in potassium solutions, up to 120 min at 12°C in a solution of 100 mM KCl. The prolonged duration of movement might be caused by reactivation of sperm or gradual activation of sperm motility. Calcium had an inhibitory effect on sperm motility in muskellunge, starting at 3 mM CaCl2 with 30 mM tris–HCl at pH 8.0. Semen diluted in calcium-supplemented solutions did not disperse well, and the sperm tended to form clumps. The mechanism involved in muskellunge sperm motility control markedly differs from that in salmonids (inhibitory function of K+ and activatory role of Ca2+) and cyprinids (no effect of Ca2+).