Difference In Maximal Response Research Articles

High-throughput evaluation of epilepsy-associated KCNQ2 variants reveals functional and pharmacological heterogeneity.

Hundreds of genetic variants in KCNQ2 encoding the voltage-gated potassium channel KV7.2 are associated with early onset epilepsy and/or developmental disability, but the functional consequences of most variants are unknown. Absent functional annotation for KCNQ2 variants hinders identification of individuals who may benefit from emerging precision therapies. We employed automated patch clamp recordings to assess at, to our knowledge, an unprecedented scale the functional and pharmacological properties of 79 missense and 2 inframe deletion KCNQ2 variants. Among the variants we studied were 18 known pathogenic variants, 24 mostly rare population variants, and 39 disease-associated variants with unclear functional effects. We analyzed electrophysiological data recorded from 9,480 cells. The functional properties of 18 known pathogenic variants largely matched previously published results and validated automated patch clamp for this purpose. Unlike rare population variants, most disease-associated KCNQ2 variants exhibited prominent loss-of-function with dominant-negative effects, providing strong evidence in support of pathogenicity. All variants responded to retigabine, although there were substantial differences in maximal responses. Our study demonstrated that dominant-negative loss-of-function is a common mechanism associated with missense KCNQ2 variants. Importantly, we observed genotype-dependent differences in the response of KCNQ2 variants to retigabine, a proposed precision therapy for KCNQ2 developmental and epileptic encephalopathy.

Validation of an 8-minute Self-Paced Graded Exercise Testing Protocol to Elicit Maximal Responses

Recent examinations have turned to the development and validation of a 10-min self-paced graded exercise testing protocol. The 10-min duration was chosen because it represents the mean value of the recommended 8-12min ideal protocol duration; however, literature exists suggesting that an 8min duration may be more appropriate to elicit maximal exercise responses. Furthermore, a gender effect may exist to explain the finding. PURPOSE: We sought to examine maximal exercise responses during an 8-min self-paced (8SPV), 10-min self-paced (10SPV) and standardized graded exercise testing (GXT) treadmill protocols. METHODS: Sixteen recreationally active males (n=8) and females (n=8) completed three separate tests in a randomized order: a) 8SPV consisting of eight 1-min stages of increasing speed clamped by the Borg RPE6-20 scale, b) 10SPV consisting of five 2-min stages clamped of increasing speed clamped by the Borg RPE6-20 scale, c) traditional Bruce protocol as the GXT. 8SPV and 10SPV maintained a 3% grade. A two-way (gender x protocol) ANOVA with repeated measures was employed to examine differences in maximal responses between protocols. Paired samples t-tests were used to examine the difference in maximal velocity between 8SPV and 10SPV. RESULTS: No gender effects were revealed. Maximal values for 8SPV, 10SPV and GXT were similar (p > 0.05) for oxygen consumption (48.0±5.6; 51.8±10.5; 51.4±7.5 mL•kg-1•min-1), heart rate (193±11; 195±9; 194±13 beats•min-1), respiratory exchange ratio (1.11±0.07; 1.12±0.06; 1.13±0.05), ventilation (103.2±22.4; 113.0±30.3; 112.3±33.0 L•min-1), respectively. Maximal velocity for 8SPV and 10SPV were also similar (15.1±3.1 vs. 14.6±2.7 km•hr-1, p > 0.05). CONCLUSION: Given no differences between protocols, 8SPV may serve as a valid and time efficient option to elicit maximal responses during self-paced exercise in recreationally trained college-aged men and women.

Myogenic regional responsiveness to cholinergic and vipergic stimulation in human colon

Differences in the actions of enteric neurotransmitters on colonic circular and longitudinal muscle layers have not been clearly determined, nor the possible existence of intrinsic myogenic phenotypes that might contribute to regional differences in human colon motor activity. The aim of this study was to analyze the direct pharmaco-mechanical coupling of carbachol (CCh) and vasoactive intestinal polypeptide (VIP) on human colonic smooth muscle strips and cells. Circular and longitudinal muscle strips and cells were obtained from 15 human specimens of ascending and sigmoid colon. Both isometric tension on muscle strips and contraction and relaxation on cells were measured in response to increasing CCh and VIP concentrations. Circular muscle strips of ascending colon were more sensitive to the effect of CCh than that of sigmoid colon, EC(50) values being, respectively, 4.15μmolL(-1) and 8.47μmolL(-1) (P<0.05), although there were no differences in maximal responses. No regional differences were observed in longitudinal muscle strips or in smooth muscle cells. Maximal responses to CCh were higher on circular than longitudinal muscle strips and cells throughout the colon. A greater sensitivity to VIP was observed in ascending colon compared with sigmoid colon, both in circular (EC(50:) 0.041 and 0.15μmolL(-1) , respectively, P<0.01) and longitudinal (EC(50:) 0.043 and 0.09μmolL(-1) , respectively, P<0.05) strips, and similar differences were observed in longitudinal smooth muscle cells (EC(50:) 44.85 and 75.24nmolL(-1) , respectively, P<0.05). Regional myogenic differences in pharmaco-mechanical coupling between the enteric neurotransmitters and smooth muscle contribute to the complex regional motor patterns of human colon.

Impact of the Exercise Mode on Exercise Capacity: Bicycle Testing Revisited

To test the performance of a tool designed to estimate functional capacity in order to select a bicycle ramp protocol yielding a test duration from 8 to 12 min in healthy individuals, and to assess differences in exercise responses between bicycle and treadmill tests. Forty-one healthy and physically active volunteers (10 women; median age, 37 years; interquartile range [IQR], 29.5 to 41 years) performed an individualized ramp exercise protocol on a bicycle ergometer and a treadmill in random order. Prior to testing, the Veterans Specific Activity Questionnaire (VSAQ) combined with a modified variant of the VSAQ nomogram (metabolic equivalents [METs] derived from VSAQ and age with the modified nomogram, resulting in METs nomogram) was used to estimate exercise capacity and to select the treadmill protocol. The corresponding bicycle work capacity nomogram (in watts) was derived by the following equation: (METs nomogram - 1) x body weight/3.486. Using treadmill tests, all 41 participants (100%) achieved maximal exercise from 8 to 12 min, as compared to 33 participants (80%) for the bicycle tests (p = 0.003). Peak oxygen uptake (V(O2)) [bicycle: median, 49.7 mL/kg/min (IQR, 45.4 to 56.6 mL/kg/min); vs treadmill: median, 53.1 mL/kg/min (IQR, 47.3 to 57.7 mL/kg/min; p < 0.0001)] was lower using the bicycle compared to the treadmill. However, the difference in peak V(O2) values between the two exercise modes was lower (2.6 mL/kg/min; IQR, 1.1 to 4.3 mL/kg/min), corresponding to 4.6% (IQR, 2.4 to 8.5%) of the lower of both values than reported in previous studies, and seven participants (17%) even achieved a higher peak V(O2) using the bicycle. A modified version of the VSAQ can be effectively used to select appropriate ramp rates for exercise testing using a bicycle ergometer in healthy individuals. Differences in maximal responses are less than those previously reported, suggesting that the bicycle ergometer is an appropriate alternative to the treadmill test in healthy volunteers.

MECHANISMS UNDERLYING RELAXATION OF RABBIT AORTA BY BAY 41‐2272, A NITRIC OXIDE‐INDEPENDENT SOLUBLE GUANYLATE CYCLASE ACTIVATOR

1. The compound BAY 41-2272 (5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine) has been described as a potent, nitric oxide (NO)-independent, stimulator of soluble guanylate cyclase. In the present study, the mechanisms underlying the relaxant effect of BAY 41-2272 in endothelium-intact and -denuded precontracted rabbit aortic rings were investigated. 2. Male New Zealand white rabbits were anaesthetized with pentobarbital sodium. Aortic rings were transferred to 10 mL organ baths containing oxygenated and warmed Krebs' solution. Tissues were connected to force-displacement transducers and changes in isometric force were recorded. Aortic rings were precontracted submaximally with phenylephrine (1 micromol/L). 3. The addition of BAY 41-2272 (0.01-10 micromol/L) to the organ bath produced concentration-dependent relaxations of the aortic rings with a higher potency in endothelium-intact (pEC50 6.59 +/- 0.05) compared with endothelium-denuded (pEC50 6.19 +/- 0.04; P < 0.05) preparations. No differences in maximal responses were observed in either preparation. The NO synthesis inhibitor NG-nitro-L-arginine methyl ester (100 micromol/L) produced a 2.1-fold rightward shift in endothelium-intact (P < 0.01) rings, but had no effect in endothelium-denuded rings. The soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 1 micromol/L) caused significant rightward shifts of the concentration-response curves to BAY 41-2272 of 4.9- and 2.6-fold in endothelium-intact and -denuded rings, respectively. The phosphodiesterase-5 inhibitor sildenafil (0.1 micromol/L) significantly potentiated the relaxant effects of BAY 41-2272 in both endothelium-intact and -denuded rings. 4. At 1 micromol/L, BAY 41-2272 significantly elevated the aortic cGMP content above basal levels in both endothelium-intact and -denuded rings. Furthermore, ODQ reduced BAY 41-2272-elicited increases in cGMP content by 17 and 90% in endothelium-intact and -denuded rings, respectively (P < 0.01). 5. In conclusion, BAY 41-2272 potently relaxes endothelium-intact and -denuded rabbit aortic rings. The basal release of endothelium-derived NO enhances BAY 41-2272-induced relaxations, suggesting a synergistic effect of BAY 41-2272 and NO on soluble guanylate cyclase. In addition, the endothelium-independent relaxation involves both GMP-dependent and -independent mechanisms.

Early Gestation Dexamethasone Exposure Alters Coronary Artery Reactivity in Newborn Lambs.

BACKGROUND: Exposure of the ovine fetus to glucocorticoids early in gestation programs postnatal elevation of blood pressure and enhanced coronary artery reactivity to second messenger-dependent vasoconstrictors. It is unknown whether the coronary alterations are a direct consequence of the corticosteroid exposure or related to the hypertension that evolves over the first 4 months of life. HYPOTHESIS: Early gestation glucocorticoid exposure enhances newborn lamb coronary artery vascular reactivity in the absence of systemic hypertension. METHODS: Dexamethasone (dex, 0.28 mg/kg/day iv for 48 hours) was administered to pregnant ewes at 27–28 days gestation (term being 145 days). The ewes were allowed delivery, and offspring were studied at a postnatal age of 8 ± 2 days (N = 6). Non-dex exposed age-matched control lambs were used for all comparisons (N = 6). Vascular catheters were placed 48 h prior to recording blood pressures. The contractile responses of circumflex coronary, mesenteric and femoral artery rings were then measured by wire myography. RESULTS: Exposure to dex was not associated with alterations in mean arterial blood pressure or heart rate at 8 days of life. Coronary vessels from dexamethasone-exposed sheep exhibited enhanced vasoconstriction to endothelin-1 and acetylcholine (both P < 0.05). There was no difference in maximal response of the coronary arteries to potassium chloride or angiotensin II. Dex exposure was associated with attenuation in vasodilatation to adenosine, but not sodium nitroprusside or forskolin. No differences in contractile response were detected between dex-exposed and control mesenteric or femoral arteries. CONCLUSION: Early gestation glucocorticoid exposure selectively programs postnatal alterations in coronary artery vascular reactivity prior to the development of hypertension. These findings suggest coronary artery dysfunction is a primary programming phenomenon and not secondary to alterations in blood pressure. These coronary vascular alterations may provide a mechanistic link between an adverse intrauterine environment and later coronary artery dysfunction.

RAT RENAL FUNCTION FOUR DAYS AFTER BILE-DUCT LIGATION: EFFECTS OF INDOMETHACIN AND VASOACTIVE AGENTS

The purpose of this study was to assess the effects of the cyclooxygenase inhibitor, indomethacin, and some vasoactive agents on the renal functional parameters during the early liver injury induced by four days bile duct ligation (BDL). Wistar rats with four days-BDL and control-sham operated were used. Renal function was measured in anesthetized rat treated with a single dose of indomethacin (control, 0.3, 1.0, 3.0 mg/kg b.w.; i.v.) one hour before clearance studies. Sulindac effects were also evaluated (5 mg/kg b.w., i.p). Isolated rat kidney preparations from control and BDL donor rats were used to study renal vascular response to noradrenaline, carbachol or sodium nitroprusside. The bile duct ligation promoted a diminished renal cortical plasma flow (RCPF) on the fourth day post surgery accompanied with a diminution in the glomerular filtration rate (GFR), increased filtration fraction and increased fractional excretion of water and sodium. Indomethacin 0.3 mg/kg induced an increase in GFR and RCPF, maintaining the high filtration fraction in BDL rats. The other doses did not alter these parameters as compared with bile duct ligated rats without treatment, but indomethacin 3 mg/kg caused a significant increase in filtration fraction. Indomethacin induced dose-dependent diminution in natriuresis in sham and BDL groups. Sulindac did not modify hemodynamic parameters, but induced antinatriuresis and antidiuresis in both experimental groups. Maximal vascular responses to noradrenaline measured in isolated rat kidneys were statistically diminished in BDL-rats as compared with controls (C, n = 7; 35.0 ± 2.3 mmHg ml−1 min; BDL-rats, n = 5; 23.8 ± 0.7 mmHg ml−1 min; p<0.02), without changes in EC15. Maximal relaxation induced by sodium nitroprusside in the phenylephrine (PHE)-pre-constricted renal vasculature in control preparations did not differ from that observed in BDL group (C: n = 6; 49.5 ± 2.3%). Values of EC50 were 1.26 ± 0.07 µM (n = 6) in control preparations and 0.34 ± 0.03 µM (n = 4) in kidneys from BDL-rats (p<0.001). Carbachol induced a biphasic relaxation of PHE-pre-constricted renal vasculature. No differences in maximal responses were found. EC50 value of the second phase in BDL group was significantly decreased compared to control preparations (C: n = 6, 0.47 ± 0.05 µM; BDL: n = 6, 0.22 ± 0.03 µM; p<0.001). The present results show that the altered renal function after a short time post bile duct ligation is determined, at least in part, by increased release of arachidonic derivatives in vascular bed and tubular cells. At this stage of liver injury, the alteration in the renal vascular response to different vasoactive agents is remarkable.

Differences in sensitivity, maximal response and position of the concentration-response curve to methacholine between asthmatics, patients with allergic rhinitis and healthy subjects

The aim of this study was to detect differences in maximal response and position of the concentration-response curves to methacholine between asthmatics and subjects with allergic rhinitis. A total of 228 adults (107 mild asthmatics, 96 allergic rhinitics and 25 healthy control subjects) were challenged with methacholine. The test was interrupted when FEV 1 dropped by more than 40% or when the highest concentration of methacholine (200 mg ml −1) had been administered. Concentration-response curves were characterized by their PC 20 (concentration of methacholine that produced 20% fall in FEV 1=airway sensitivity), and if possible, by their EC 50 (concentration of methacholine that produced 50% of the maximal response=position) and level of plateau. The proportion of subjects with plateau was significantly lower in asthmatics (18·7%) than in either allergic rhinitics (57·3%) or healthy subjects (92%). It was also significantly lower in allergic rhinitics than in healthy subjects. The level of plateau for asthmatics was (means ± SD) 31·5 ± 5·5%, compared with 20·8 ± 8·1% in allergic rhinitics and 13·7 ± 6·7% in healthy subjects ( P<0·01). It was also higher in allergic rhinitics than in healthy subjects ( P<0·01). The EC 50 values were decreased in asthmatics when they were compared with either allergic rhinitics or healthy subjects (geometric mean EC 50: asthmatics=2·7 mg ml −1, allergic rhinitics=6·2 mg ml −1, healthy subjects=8·7 mg ml −1; P<0·01), but no significant differences were detected between allergic rhinitics and healthy subjects. These results demonstrate that in subjects with allergic rhinitis, the prevalence and level of the plateau on the methacholine concentration-response curve is intermediate between that of asthmatics and normals. Furthermore, while the asthmatic curves differ from normal in having both an increased maximal response and a leftward shift, the rhinitic curves differ only in terms of plateau level. These results suggest that airway responsiveness in asthma and allergic rhinitis may be a consequence of mechanisms that are at least partially different.

In vivo and in vitro evidence of altered nitric oxide metabolism in the spontaneously diabetic, insulin‐dependent BB/Edinburgh rat

1. Altered vasoreactivity may contribute significantly to the pathogenesis of diabetic vascular complications. This study investigated the effect of (a) insulin-related diabetes, and (b) chronic in vivo administration of N(omega)-nitro-L-arginine ester (L-NAME), a nitric oxide (NO) synthase inhibitor, on mean arterial pressure and in vitro vascular reactivity to noradrenaline in mesenteric arterial bed preparations from spontaneously diabetic, insulin-dependent and treated BB rats, the best animal model of insulin-dependent mellitus (IDDM) currently available. Four groups of animals from the Edinburgh colony (BB/E) of spontaneous diabetic BB rats were studied: age-matched (mean +/- s.e. mean = 156 +/- 2d) non-diabetic (glycated haemoglobin = 3.8 +/- 0.1%) and insulin-treated diabetic (glycated haemoglobin = 6.2 +/- 0.5%; duration of diabetes = 56 +/- 4 d) groups were either L-NAME treated (oral dose = 27 +/- 1 mg kg-1 d-1; duration of treatment from 30 until 153 days of age) or untreated. Although our diabetic BB/E rats do not achieve overall normoglycaemia, individual adjustment of the daily insulin dose administered to every diabetic rat achieves better glycaemic control than previous groups studying altered vascular reactivity and endothelial dysfunction in this animal model of diabetes. 2. Mean arterial pressure (measured directly via indwelling carotid arterial cannulae) was not significantly different between non-diabetic (116 +/- 3 mmHg; n = 10) and diabetic (122 +/- 2 mmHg; n = 12) BB/E rats. L-NAME treatment significantly (P < 0.001) increased mean arterial pressure in both groups (165 +/- 6 mmHg; n = 9 and 142 +/- 4 mmHg; n = 6 respectively) but the degree of hypertension observed in L-NAME-treated diabetic rats was significantly (P < 0.01) attenuated compared to non-diabetic rats treated with L-NAME. 3. Mesenteric arterial bed preparations were cannulated under anesthesia, excised and intralumenally perfused ex vivo with noradrenaline (0.2-20 microM). Basal perfusion pressures were not significantly different in mesentery preparations from non-diabetic (27.0 +/- 2.6 mmHg) and diabetic (27.1 +/- 3.2 mmHg) BB/E rats. There was no significant difference in maximal response above basal perfusion pressure (MAX) or pEC50, defined as the negative log of the agonist concentration required to give 50% of the maximal response above basal perfusion pressure, to noradrenaline in untreated non-diabetic (166 +/- 7 mmHg and 5.74 +/- 0.05 respectively) and diabetic (170 +/- 11 mmHg and 5.59 +/- 0.05) BB/E rats. 4. In vivo treatment of non-diabetic and diabetic BB/E rats with L-NAME had no significant effect on basal perfusion pressure (25.9 +/- 4.3 mmHg and 28.5 +/- 3.9 mmHg respectively). L-NAME treatment in vivo increased (P < 0.001) MAX to noradrenaline of non-diabetic rats (224 +/- 8 mmHg) but did not affect the value for diabetic rats (178 +/- 14 mmHg). L-NAME treatment did not alter after the pEC50 values in either group (5.71 +/- 0.05 and 5.65 +/- 0.05). 5. Consistent with previous studies using vascular preparations from spontaneously diabetic BB rats, mesentery preparations from diabetic BB/E rats (n = 12) exhibited a significantly reduced vasodilator response to acetylcholine (F value = 4.4, P < 0.05) across the concentration range studied compared to non-diabetic BB/E rats (n = 12) although there was no significant difference in maximal relaxation (diabetic 53.1 +/- 4.3% vs non-diabetic 55.7 +/- 5.5%) or pEC50, (diabetic 6.92 +/- 0.25 vs non-diabetic 7.49 +/- 0.22). There was no significant (F value = 0.8, P > 0.1) difference in the response to GTN between preparations from non-diabetic and diabetic rats (maximal relaxation: 49.6 +/- 3.7% vs 48.5 +/- 4.3%; pEC50: 7.84 +/- 0.12 vs 7.89 +/- 0.22 respectively). 6. In conclusion, vascular responsiveness to noradrenaline is not impaired in spontaneously diabetic BB/E rats with significantly better glycaemic control than those used in previous studies. However, following chronic L-NAME treatment, diabetic BB/E rats exhibit attenuated hypertension and an absence of enhanced vascular responsiveness to noradrenaline in vitro compared to similarly treated non-diabetic rats. These results, together with the significantly impaired endothelium-dependent vasodilatation and unchanged endothelium-independent vasodilatation in vitro of preparations from diabetic BB/E rats, are consistent with the hypothesis that functional changes in the synthesis and metabolism of NO (rather than altered vascular responsiveness to NO) occur in diabetes. Our results indicate that good glycaemic control alone is insufficient to prevent these abnormalities in NO availability and further studies to characterize the origin of these changes are necessary.

Pharmacological analysis of dopamine stimulation of [35S]-GTP gamma S binding via human D2short and D2long dopamine receptors expressed in recombinant cells.

1. The activation of G-proteins by agonist-occupied D2 or D3 dopamine receptors in membranes from recombinant cells expressing the cloned receptors has been analysed by a [35S]-guanosine 5'-[gamma-thio] triphosphate ([35S]-GTP gamma S) binding assay. 2. The rate of [35S]-GTP gamma S binding was increased by dopamine in a dose-dependent manner in membranes from CHO cells stably expressing either the D2short or D2long dopamine receptor. 3. The dopamine-induced stimulation of [35S]-GTP gamma S binding could be inhibited by a range of antagonists. Affinities for antagonists derived from the inhibition of the dopamine stimulation of [35S]-GTP gamma S binding correlated very well with affinities derived from radioligand binding studies. 4. When the maximum [35S]-GTP gamma S binding responses stimulated by dopamine acting at different receptor subtypes were compared, there was a tendency for the stimulation via the D2short receptor to be greater than via the D2long receptor and for the stimulation via the D3 dopamine receptor to be less than for either D2 receptor. These differences in maximal response were also seen when the inhibitory effects of dopamine on adenylyl cyclase via the three receptor subtypes were compared. 5. The stimulation of [35S]-GTP gamma S binding by dopamine in membranes from recombinant cells therefore provides an excellent system for studying the molecular nature of agonism and the receptor/G-protein interactions for these receptors.

Neonatal capsaicin treatment alters basal pulmonary mechanics and response to methacholine in F344 rats

Full methacholine dose-response curves were performed on anesthetized tracheostomized Fischer 344 adult rats treated neonatally with capsaicin (50 mg/kg) or with vehicle alone. Capsaicin, the hot extract of pepper, releases substance P (SP) from nonmyelinated sensory nerve endings and causes acute bronchoconstriction and airway microvascular leakiness. Chronic treatment with capsaicin leads to depletion of SP and other tachykinins from afferent C-fibers and can therefore be used as a tool to investigate the contribution of SP innervation to airway responses. The rats (9 controls and 6 treated with capsaicin) were paralyzed with succinylcholine and mechanically ventilated at a constant tidal volume and frequency. Airway resistance (RL) and dynamic compliance (Cdyn) were determined at each dose of methacholine from measurements of volume, flow, and transpulmonary pressure. Capsaicin-treated rats were found to have a significantly reduced baseline RL [0.150 +/- 0.039 (SD) vs. 0.225 +/- 0.050 cmH2O.ml-1.s, P = 0.009] and a correspondingly significantly elevated Cdyn (0.371 +/- 0.084 vs. 0.268 +/- 0.053 ml/cmH2O, P = 0.012). There was no significant difference in sensitivity to methacholine, but the maximal response to methacholine was significantly greater in the capsaicin-treated rats. In terms of RL, the maximal response for capsaicin-treated rats was 6.03 x baseline +/- 0.98 vs. 4.30 x baseline +/- 1.80 (P = 0.05) for controls, and for Cdyn changes the maximal decrease was 5.75 x baseline +/- 1.22 vs. 3.83 +/- 0.69 (P = 0.002). The observed differences in RL and Cdyn coupled with the differences in maximal responses can be attributed to the selective destruction of a subpopulation of pulmonary afferent C-fibers.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium antagonists inhibit contractions to norepinephrine in the rat aorta, in the absence, but not in the presence of the endothelium

1. 1. To compare the effect of diltiazem and verapamil on the responsiveness of vascular smooth muscle to norepinephrine in the presence and absence of the endothelium, rings of rat aorta were studied in organ chambers. 2. 2. The removal of the endothelium decreased the ED 50 to norepinephrine and augmented the maximal response to the catecholamine. 3. 3. The contraction to norepinephrine consisted of a rapid initial (phasic) and a (tonic) part. The tonic part was reduced in the presence of the endothelium. 4. 4. Diltiazem shifted the concentration-response curve to norepinephrine to the right only in rings without endothelium and reduced the difference in maximal response between rings with and without endothelium. 5. 5. Verapamil abolished the difference in sensitivity (ED 50) between rings with and without endothelium. 6. 6. Oxyhemoglobin prevented the inhibitory effect of the endothelium on the response to norepinephrine, and unmasked a shift of the ED 50 to the catecholamine to the right by diltiazem in rings with endothelium. 7. 7. These experiments suggest that spontaneously released endothelium-derived relaxing factor(s) is a functional antagonist of norepinephrine-induced contractions, presumably by reducing the stimulated influx of extracellular Ca 2+.

Multihormonal regulation and kinetics of induction of a hepatic mRNA sequence which is slowly responsive to triiodothyronine.

In contrast to the rapid response of mRNA-S14 which occurs within 20 min after L-triiodothyronine (T3) administration, the induction of other rat hepatic mRNA sequences exhibits a lag time of several hours. We have studied the induction of mRNA-S11 which codes for a protein of pI 6.1 and an Mr of 22,500 as a model of such a slowly responsive gene. In addition to T3, both glucocorticoids and growth hormone regulate the expression of this gene. For each of these stimuli, the response exhibits a lag time of approximately 6 h. Following this lag time, there is a linear increase in the level of mRNA-S11 induced by a single maximal dose of T3, dexamethasone, and growth hormone to levels of 15-, 6-, and 3-fold, respectively, in excess of the hypothyroid base-line levels. The similarity in the lag time and the differences in maximal responses effectively argues against the possibility that the effect of one hormone is mediated exclusively by a change in the secretion or metabolism of another. Support for a direct action of T3 and glucocorticoids on the hepatic cell comes from the observation that these hormones stimulate mRNA-S11 in rat hepatocytes under primary culture. The increases in in vitro nuclear transcription measured following T3 and dexamethasone administration were clearly insufficient to account for the observed increases in mRNA. Furthermore, the hormonal induction of mRNA-S11 was promptly abrogated by cycloheximide (10 mg/kg) injected 6 h after the administration of T3 or dexamethasone at the time of the expected mRNA increase. The post-transcriptional control of the mRNA-S11 and its sensitivity to cycloheximide are similar to previously documented responses of the rapidly induced mRNA-S14 and suggest for both sequences a requirement for ongoing synthesis of rapidly turning over proteins. We speculate that the lag time of response of a given gene to T3 or glucocorticoid may be an intrinsic characteristic of the gene and may represent a common set of molecular events involved in the activation of that gene by diverse stimuli.

Extracellular calcium and altered vascular responsiveness in the deoxycorticosterone acetate-salt rat.

This study investigated the effects of altered extracellular Ca2+ on in vitro femoral arterial smooth muscle responsiveness in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Compared with controls, femoral arteries from DOCA-salt rats showed a significant increase in sensitivity to KCl and norepinephrine in normal Ca2+ (2.5 mM). Although no difference in maximal contractile response to KCl was observed between groups, there was a significant difference in maximal response to norepinephrine. Dose-response curves in low Ca2+ (0.25 mM) resulted in a significant decrease in the sensitivity of femoral arteries from DOCA-salt rats to KCl and NE so that the responses were similar to those of controls. Relaxation of femoral arteries from DOCA-salt rats after washout of the KCl contraction was significantly slower than that of controls in both low and normal Ca2+. Isoproterenol-induced relaxation of femoral arteries from DOCA-salt rats was significantly attenuated in normal Ca2+. Sensitivity of femoral arteries from DOCA-salt rats to isoproterenol increased in low Ca2+, but maximal relaxation was unaltered. Whereas no difference in maximal relaxation to NaNO2 was seen in femoral arteries from either group in normal Ca2+, a significant decrease in sensitivity to NaNO2 was observed in femoral arteries from DOCA-salt rats. In low Ca2+ the response of femoral arteries from DOCA-salt rats to NaNO2 was similar to that of controls. These results suggest that the increased vascular smooth muscle responsiveness to KCl and norepinephrine seen in DOCA-salt hypertension is due to increased sensitivity of the vascular smooth muscle to Ca2+. Extracellular Ca2+, however, plays only a minor role in the decreased vasodilator responsiveness seen in this form of hypertension.