Physiologic effect of nifedipine and tamsulosin on contractility of distal ureter.

BACKGROUND AND PURPOSE: Renal colic not only leads to significant morbidity but imposes a financial burden on society in lost productivity and healthcare dollars spent. Both tamsulosin and nifedipine can facilitate spontaneous stone passage in the distal ureter and reduce the associated colic. We evaluated the physiologic effect of these agents on the distal pig ureter. MATERIALS AND METHODS: Bilateral ureters were removed en bloc with the bladder trigone from three pigs. Five-millimeter rings were taken from the intramural and distal ureter. Isometric tension recording was performed during both spontaneous and electrically stimulated contraction. Measurements of contractile interval and amplitude were taken in baseline buffer solution, nifedipine at 10(-6) g/L, and tamsulosin at 10(-6) g/L. Spontaneous contractile activity was also measured with phenylephrine at 10(-4) g/L followed by tamsulosin at 10(-6) g/L. RESULTS: Under conditions of spontaneous contractility, phenylephrine decreased the contraction interval by 46%, an effect which was reversed by tamsulosin. Tamsulosin increased the baseline interval by 27% (P < 0.025) and decreased the amplitude by 7% (P > 0.1). Nifedipine blocked all contractile activity. Under stimulated contractility, tamsulosin had no effect on the interval and decreased the amplitude by 7% (P > 0.1). Nifedipine blocked all contractile activity. CONCLUSIONS: We believe that both tamsulosin and nifedipine prevent the disorganized antiperistalsis associated with ureteral spasm while allowing some degree of antegrade fluid-bolus (stone) propagation. It is this mechanism of action that facilitates spontaneous passage and reduces associated renal colic when tamsulosin and nifedipine are used for the management of ureteral stone disease.

Metabolic activation of AMP kinase in vascular smooth muscle.

AMP-activated kinase (AMPK) is a highly conserved heterotrimeric kinase that functions as a metabolic master switch to coordinate cellular enzymes involved in carbohydrate and fat metabolism that regulate ATP conservation and synthesis. AMPK is activated by conditions that increase AMP-to-ATP ratio, such as exercise and metabolic stress. In the present study, we probed whether AMPK was expressed in vascular smooth muscle and would be activated by metabolic stress. Endothelium-denuded porcine carotid artery segments were metabolically challenged with 2-deoxyglucose (10 mM) plus N(2) (N(2)-2DG). These vessels exhibited a rapid increase in AMPK activity by 1 min that was near maximal by 20 min. AMPK inactivation on return to normal physiological saline was approximately 50% in 1 min and fully recovered by 5 min. Immunoprecipitation of the alpha(1)- and alpha(2)-catalytic subunit followed by immunoblot analysis for [P]Thr(172)-AMPK indicates that alpha(1)-AMPK accounts for all activity. Little if any alpha(2)-AMPK was detected in carotid smooth muscle. AMPK activity was not increased by contractile agonist (endothelin-1) or by the reported AMPK activators 5-aminoimidazole-4-carboxamide ribofuranoside (2 mM), metformin (2 mM), or phenformin (0.2 mM). AMPK activation by N(2)-2DG was associated with a rapid and pronounced reduction in endothelin-induced force and reduced phosphorylation of Akt and Erk 1/2. These data demonstrate that AMPK expression differs in vascular smooth muscle compared with striated muscles and that activation and inactivation after metabolic stress occur rapidly and are associated with signaling pathways that may regulate smooth-muscle contraction.

High-fat diet alters K+-currents in porcine coronary arteries and adenosine sensitivity during metabolic inhibition.

Coronary arteries from animals on normal diets (ND) exhibit well-maintained responses to dilators under ischemic conditions. The reported altered metabolic requirements and K+-currents in blood vessels from hypercholesterolemic animals fed high-fat diets (HF) led us to hypothesize that under metabolically depressed conditions (N2/2-deoxyglucose) coronary arteries from pigs would exhibit significantly decreased responses to adenosine (ADO) as compared with pigs given ND. Diet had no major effect on responses of coronary rings to ET-1, nor on the sensitivity to ADO or 2-chloroadenosine (2-CAD) relaxation under metabolically supported conditions. During metabolic inhibition the response curves for both ADO and 2-CAD were shifted to the right (P < 0.05), with the HF group shifted about 4-fold more than ND (P < 0.05). To determine the involvement of K+-channels, ADO responses were measured in the presence of 4-aminopyridine (4-AP, 1 mM) or glybenclamide (GLYB, 10 microM). The larger shift in the HF group during metabolic inhibition was not affected by GLYB, but disappeared in the presence of 4-AP with ND now behaving similarly to HF. These results indicate that HF diet may have a 4-AP-like effect on voltage-dependent K+-channels (KV). Patch-clamp measures of whole cell K- currents showed the HF cells to have reduced 4-AP sensitive currents (P < 0.02). The 4-AP insensitive currents were similar in both groups. Thus, reduced KV channel activity may play a role in the depressed ADO relaxation associated with metabolic inhibition of HF coronary arteries. These factors may place the coronary circulation of HF at increased risk during an ischemic episode.

Selective transport of adenosine into porcine coronary smooth muscle.

Adenosine (ADO), an endogenous regulator of coronary vascular tone, enhances vasorelaxation in the presence of nucleoside transport inhibitors such as dipyridamole. We tested the hypothesis that coronary smooth muscle (CSM) contains a high-affinity transporter for ADO. ADO-mediated relaxation of isolated large and small porcine coronary artery rings was enhanced 12-fold and 3.4-fold, respectively, by the transport inhibitor, S-(4-nitrobenzyl)-6-thioinosine (NBTI). Enhanced relaxation was independent of endothelium and was selective for ADO over synthetic analogs. Uptake of [(3)H]ADO into freshly dissociated CSM cells or endothelium-denuded rings was linear and concentration dependent. Kinetic analysis yielded a maximum uptake (V(max)) of 67 +/- 7.0 pmol. mg protein(-1). min(-1) and a Michaelis constant (K(m)) of 10. 5 +/- 5.8 microM in isolated cells and a V(max) of 5.1 +/- 0.5 pmol. min(-1). mg wet wt(-1) and a K(m) of 17.6 +/- 2.6 microM in intact rings. NBTI inhibited transport into small arteries (IC(50) = 42 nM) and cells. Analyses of extracellular space and diffusion kinetics using [(3)H]sucrose indicate the V(max) and K(m) for ADO transport are sufficient to clear a significant amount of extracellular adenosine. These data indicate CSM possess a high-affinity nucleoside transporter and that the activity of this transporter is sufficient to modulate ADO sensitivity of large and small coronary arteries.

Size dependence of functional alterations in mesenteric arteries from the aldosterone-salt hypertensive rat.

We tested the hypothesis that an elevated potassium-42 ((42)K(+)) efflux (highly dependent on Ca(2+)) and an increase in the sensitivity of contraction and (42)K(+) efflux to norepinephrine (NE) in conduit arteries of aldosterone-salt hypertensive rats (AHR) extended to smaller, distributing arteries. Functional endpoints were compared in two sizes of arteries from the mesenteric bed: second-order branches of the superior mesenteric artery (SMA branches) and the SMA. Contraction and free cytosolic Ca(2+) concentration ([Ca(2+)](c); fura-2 microfluorometry) were measured simultaneously and (42)K(+) efflux was measured separately in SMA branches. Contraction and (42)K(+) efflux were measured separately in SMA. Basal tone, [Ca(2+)](c), and (42)K(+) efflux were similar in SMA branches from AHR and control-salt rats (CSR). However, basal (42)K(+) efflux was elevated in SMA from AHR compared to CSR. The sensitivity of the contractile, [Ca(2+)](c), and (42)K(+) efflux responses to NE was similar in SMA branches. In contrast, the sensitivity of the contractile and (42)K(+) efflux responses to NE was enhanced in SMA from AHR compared to CSR. Inhibiton of endothelium-derived vasoactive substances by pretreatment with N(omega)-nitro-L-arginine methyl ester and indomethacin significantly shifted the NE concentration-response relationships to the left for contraction, [Ca(2+)](c), and (42)K(+) efflux in both SMA branches and SMA from CSR. A similar shift to the left was observed in AHR for contraction but not consistently for [Ca(2+)](c) and (42)K(+) efflux. We conclude that SMA branches from AHR demonstrate neither the elevated basal (42)K(+) efflux, nor the NE supersensitivity exhibited by SMA. Endothelial function was not impaired both in SMA and SMA branches.

Endothelin-1 sensitivity of porcine coronary arteries is reduced by exercise training and is gender dependent.

We tested the hypothesis that exercise training reduces the sensitivity of coronary smooth muscle to endothelin-1 (ET-1), with the adaptation being greater in male than in female miniature swine. The efficacy of training was similar in males and females. Cumulative ET-1 contractile responses of coronary branches and left circumflex artery were significantly shifted to the right in exercise-trained (Ex) males but not in Ex females. Analyses of the excitatory concentration causing a 50% response (EC(50)) showed a 1.7- to 2.2-fold shift in Ex males with no change in maximum tension. Nonselective blockade of K-channel activity with tetraethylammonium (TEA; 30-50 mM) significantly shifted the EC(50) to a lower concentration in both Ex males (1.25-fold) and Ex females (2.2-fold) but not in sedentary (Sed) groups. Females (combined Sed and Ex) exhibited a greater response to TEA than did combined Sed and Ex males. Changes in [(32)P]phosphatidic acid ([(32)P]PA) provided an indicator of ET-1-induced phospholipase activity. The magnitude of the [(32)P]PA response was reduced by Ex in both males and females without affecting the EC(50). It is concluded that the contractile sensitivity of coronary arteries to ET-1 is influenced by physical activity in a gender-dependent manner. It is unclear why the contractile sensitivity in females was not reduced by Ex as in the males, because Ex significantly affected responses to TEA and ET-1 stimulation of [(32)P]PA production in both males and females. A potential gender difference in K-channel function may contribute to this discrepancy.

Endothelin-1 activates phospholipases and channels at similar concentrations in porcine coronary arteries.

Sensitivity of endothelin-1 (ET-1)-ion channel interactions has been proposed to exceed that of ET-1-phospholipase activation in vascular smooth muscle. We wanted to determine whether short-circuiting ion channels with staphylococcal alpha-toxin pores would shift the ET-1-force relation to the right as predicted from the above proposal. Medium size porcine coronary arteries (outer diameter 0.7-1.5 mm) were mounted on isometric force transducers. ET-1 concentration response curves were compared between intact rings and those subjected to alpha-toxin treatment with Ca buffered at 0.1 microM. The EC50 for treated rings (1.5 +/- 1.0 nM, n = 5 pigs) was similar to that for intact rings (1.9 +/- 0.4 nM). The Ca sensitivity of the alpha-toxin-treated rings (EC50 = 0.43 +/- 0.08 microM) was similar to that reported by other laboratories for intact and alpha-toxin-treated arteries and was shifted eightfold to the left by a high concentration of ET-1 (10 nM). Measurements of [32P]phosphatidic acid ([32P]PA) levels were used to evaluate phospholipase activity in intact arteries. The time courses for [32P]PA production and contraction were similar in response to high (100 nM) and to low (1 nM) ET-1. Significant increases in both steady-state contraction and [32P]PA occurred over a wide range of ET-1 concentrations tested (0.3-100 nM). Our findings support the concept that ET-1-phospholipase coupling is operative over the whole concentration range that induces contractile responses. It is suggested that both Ca entry and Ca sensitization processes are activated by ET-1 at low concentrations (

Mechanisms of altered contractile responses to vasopressin and endothelin in canine coronary collateral arteries.

BACKGROUND: Mature coronary collateral arteries are hyperresponsive to vasopressin; in contrast, contractile responses of collaterals to endothelin are attenuated. Our goal was to determine the cellular mechanisms underlying these differences in reactivity using two sizes of canine collateral arteries isolated from hearts subjected to chronic coronary occlusion. METHODS AND RESULTS: Contractile responses to vasopressin (100 mmol/L) were enhanced threefold to fourfold in near-resistance (approximately 200 microns lumen diameter) and conduit (approximately 500 microns lumen diameter) collateral arteries compared with similarly sized noncollateral coronary arteries (P < .01). In contrast, contractions of both sizes of collaterals in response to endothelin (0.01 to 30 nmol/L) were smaller than responses of size-matched noncollateral arteries (P < .05). Pretreatment with either indomethacin (5 mumol/L), a cyclooxygenase inhibitor, or NG-nitro-L-arginine methyl ester (100 mumol/L), a nitric oxide synthase inhibitor, did not alter the relative responsiveness of collateral arteries to vasopressin or endothelin compared with noncollateral arteries. Vasopressin produced greater increases of intracellular free Ca2+ (measured by use of fura-2 microfluorometry and Ca(2+)-dependent 42K+ efflux) in smooth muscle of collateral arteries than in smooth muscle of noncollateral arteries (P < .05). Surprisingly, endothelin-induced increases of Ca2+ were not different in smooth muscle of collateral and noncollateral arteries (P > .05). CONCLUSIONS: We conclude that altered contractile responsiveness of collateral arteries to vasopressin and endothelin does not result from altered synthesis/release of nitric oxide or prostaglandins. Parallel enhancement of vasopressin-mediated Ca2+ and contractile responses suggests increases in vasopressin receptor number, affinity, and/or efficiency of coupling mechanisms in collateral smooth muscle. The dissociation between endothelin-induced contractile and Ca2+ responses of collaterals indicates that the mechanisms involved in increasing Ca2+ sensitivity of contractile proteins during endothelin stimulation may be altered in collateral arteries.

Sodium nitroprusside alters Ca2+ flux components and Ca2(+)-dependent fluxes of K+ and Cl- in rat aorta.

1. Sodium nitroprusside (NP) caused both an inhibition of a noradrenaline (NA)-induced contraction and an elevation of cyclic guanosine 3',5'-monophosphate (cyclic GMP) in rat aorta. Both NP-induced responses were enhanced by the selective cyclic GMP phosphodiesterase inhibitor, M&B 22948 (2-o-propoxyphenyl-8-aza-purin-6-one, 30 microM). 2. The inhibition of a NA-induced contraction by NP was characterized by dissociating the intracellular Ca2+ release component from the extracellular Ca2+ influx component of the contraction. The transient contraction stimulated by NA in the absence of extracellular Ca2+ was inhibited by NP. Also, the slowly developed tension stimulated by NA in aortas depleted of stored Ca2+ and subsequently exposed to extracellular Ca2+ was inhibited by NP. Both components of contraction were equally sensitive to NP. 3. NA stimulated both unidirectional 45Ca2+ influx in the presence of extracellular Ca2+ and 45Ca2+ efflux into a 0 Ca2+ solution that contained 2 mM-ethyleneglycol-bis-(beta-aminoethylether)N,N'-tetraacetic acid (EGTA). The increased 45Ca2+ efflux is thought to reflect release of stored Ca2+ followed by membrane transport. NP greater than 10 nM inhibited both 45Ca2+ influx and release components whereas NP at 1-3 nM enhanced NA-stimulated 45Ca2+ efflux and relaxed the maintained tension caused by NA in 0 Ca2+, 2 mM-EGTA. 4. NP also inhibited the Ca2(+)-dependent 42K+ and 36Cl- effluxes from rat aorta stimulated either by NA or by high potassium. NP inhibited the contractile and flux responses to NA more effectively than the responses to high potassium. 5. These data indicate that: (1) NP reduces cytosolic Ca2+ by the combined inhibitory effects on Ca2+ influx and intracellular Ca2+ release, and by the stimulation of a Ca2(+)-ATPase; and (2) the differential sensitivity of the NA and high-potassium responses to NP may reflect underlying differences in Ca2+ handling induced by receptor occupancy and depolarization.

Depolarization-stimulated 42K+ efflux in rat aorta is calcium- and cellular volume-dependent.

The purpose of this study was to investigate the factors controlling membrane permeability to potassium of smooth muscle cells from rat aorta stimulated by depolarization. The increase 42K+ efflux (change in the rate constant) induced by depolarization (application of high concentrations of potassium chloride) was inhibited significantly by the calcium antagonists diltiazem and nisoldipine. Parallel inhibitory effects on contraction were observed. Diltiazem also inhibited potassium-stimulated 36Cl- efflux. The addition of 25-150 mM KCl to normal physiologic solution stimulated 42K+ efflux in a concentration-dependent manner. Diltiazem suppressed potassium-stimulated 42K+ efflux approximately 90% at 25 mM KCl and approximately 40% at 150 mM KCl. The ability of nisoldipine to inhibit 42K+ efflux also diminished as the potassium chloride concentration was elevated. The component of efflux that was resistant to calcium antagonists probably resulted from a decrease in the electrochemical gradient for potassium. Cellular water did not change during potassium addition. Substitution of 80 and 150 mM KCl for sodium chloride produced cellular swelling and enhanced potassium-stimulated 42K+ efflux compared with potassium chloride addition. The addition of sucrose to prevent cellular swelling reduced efflux response to potassium substitution toward that of potassium addition. A hypoosmolar physiologic solution produced an increase in the 42K+ efflux and a contracture that were both prevented by the addition of sucrose. We concluded that the depolarization-mediated 42K+ efflux has three components: one is calcium dependent; a second is dependent on cellular volume; and a third is resistant to inhibition by calcium antagonists.

Vitamin D metabolites do not alter parathyroid hormone secretion acutely.

Evidence to date has failed to show a consistent effect of vitamin D metabolites on PTH secretion. This study was undertaken to assess the possible direct, acute effects of vitamin D metabolites on PTH secretion in vitro. Ethanol has been used in several published studies as the vehicle for vitamin D metabolites. We found that 0.2-1.0% ethanol inhibited PTH release from dispersed bovine parathyroid cells (PTC). Our experiments with vitamin D metabolites used ethanol as a vehicle at a concentration less than 0.1%. When compared to ethanol treatment, 10-100 nM 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), 25 and 100 nM 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) and 100 nM 1,24,25-trihydroxyvitamin D3 (1,24,25(OH)3D3) had no effect on PTH release from PTC incubated for up to 4 h. A combination of 1,25(OH)2D3 and 24,25(OH)2D3 (each 25 or 100 nM) was without effect. Also, 100 nM 1,25(OH)2D3 had no effect on PTH release from either bovine parathyroid gland slices or from parathyroid glands from either vitamin D-replete (+D) or vitamin D-deficient (-D) rats incubated for up to 4 h. The i.v. injection of 1 microgram 1.25(OH)2D3 into -D rats had no effect on either serum PTH or calcium (Ca), either 0.5 or 1.0 h after treatment. Parathyroid glands from -D rats incubated with 0.75 mM Ca secreted more PTH than glands of similar weight from rats given 25 micrograms vitamin D3 3 days earlier, suggesting that vitamin D or a metabolite of vitamin D may modulate the sensitivity of the parathyroid gland to medium Ca. In summary, we found no evidence for a direct, acute effect of vitamin D metabolites on PTH secretion under diverse experimental conditions.

Alterations in active Na-K transport during mineralocorticoid-salt hypertension in the rat.

Studies have been conducted to determine which, if any, of the parameters governing active Na-K transport (K and/or ouabain sensitive) are altered in vascular smooth muscle during mineralocorticoid-salt hypertension. Rats with one kidney removed were treated with either aldosterone or deoxycorticosterone acetate plus saline for 3-4 wk. Ion transport was measured in arteries incubated in a physiological salt solution for periods of 4-6 h. Increased active Na efflux was observed in femoral arteries and aortas from the hypertensive group. This alteration resulted primarily from an elevation in the saturation or maximal capacity of the active transport mechanism, which operated with no significant change in cell Na concentration. The transport parameters related to ion selectivity, cooperativity, and temperature dependence were not significantly altered. Measures of active Na efflux and K influx in the same aortic strips indicated that both fluxes were elevated in the hypertensive group. The ratio of Na to K for active transport was significantly greater than one but was unchanged in the hypertensive rats. These studies provide evidence for the electrogenic operation of the Na-K pump in arterial smooth muscle and for the operation of the pump at a higher level during mineralocorticoid-salt hypertension. This alteration may result from increased turnover of individual sites or possibly the incorporation of more sites into the membrane as a result of mineralocorticoid-salt treatment.

Prolactin stimulation of parathyroid hormone secretion in bovine parathyroid cells.

Previous studies have suggested that prolactin (PRL) may affect calcium (Ca) homeostasis by an action on vitamin D metabolism. In this study, the effects of PRL on parathyroid hormone (PTH) secretion were investigated in dispersed bovine parathyroid cells (PTC). PRL (0.013-1.3 microM) caused concentration-dependent increases in PTH secretion. PRL-stimulated PTH release was apparent as early as 1 h and was progressive thereafter for up to 3 h. PRL enhanced PTH release over a wide range of ambient Ca concentrations (0.5-2.0 microM). Ovine and rat PRL were more effective than bovine PRL in stimulating PTH secretion. This effect was apparently specific for PRL because neither ovine nor bovine growth hormone stimulated PTH secretion. PRL-stimulated PTH release was not mediated through the beta-adrenergic or dopaminergic receptor systems of PTC and was not associated with increased adenosine 3',5'-cyclic monophosphate (cAMP) levels. This study demonstrated a direct effect of PRL to stimulate PTH secretion in vitro. Although these data do not provide evidence for an effect of PRL in vivo, we suggest a mechanism by which PRL may influence parathyroid function and Ca homeostasis in the bovine species.

The effects of estrogen on skeletal calcium metabolism and on plasma parameters of vitellogenesis in the male, three-toed box turtle (Terrapene carolina triunguis).

The box turtle (Terrapene carolina triunguis) does not appear to have evolved a mechanism for secondary bone formation similar to Aves inasmuch as several parameters of bone calcium metabolism were unaffected by estrogen treatment. This suggests that reproduction does not constitute a substantial stress to the calcium stores of the female, even though the box turtle is a terrestrial species for which a supply of calcium may be limited. However, the dermal bone of the shell of the box turtle appears to take up calcium at a rate equivalent to that of endoskeletal bones, suggesting that it participates in mineral homeostasis at least to the extent of other osseous elements. Calcium metabolism in the turtle may be unique among vertebrates in this respect. Estrogen treatment of male box turtles results in the appearance of a specific plasma protein in parallel with pronounced changes in plasma calcium and magnesium, suggesting the induction of vitellogenin. The magnitude of the vitellogenic response may have a seasonal component.

An improved cannulation technique for prolonged blood sampling of the American bullfrog.

A T-cannula was used to collect serial blood samples from the sciatic artery of the American bullfrog (Rana catesbeiana). The horizontal arm of the cannula was implanted in the artery and the vertical arm employed to collect blood samples. The T-cannula allowed blood flow to continue in the cannulated artery and provided long-term, repetitive blood samples from conscious, caged, but otherwise unrestrained and undisturbed animals. The cannula functioned for at least 14 days, the maximum period investigated. Athough developed for studies on amphibians and reptiles, the technique appeared to be applicable, with appropriate modifications, to a variety of small animals.