Specific binding of [3H]mepyramine to histamine H1-receptors in vascular smooth muscle membranes

The antagonist-sensitive binding of [³H]mepyramine to beef aortic membranes was as expected for binding to histamine H₁-receptors. [³H]mepyramine binds rapidly and in saturable fashion to the specific receptor sites, specific binding reaching equilibrium in 3 min at 37°CScatchard's analysis of the binding data gave a dissociation constant of 3.0 nM for the radioligand-receptor complex and maximal number of binding sites: 31 fmol/mg protein. In the competition studies histamine H₁-antagonists are more potent inhibitors of radioligand binding than H₂-antagonist. They inhibit [³H]mepyramine binding in the following order: mepyramine >triprolidine     

Histamine H1-receptors mediate phosphoinositide and calcium response in cultured smooth muscle cells-interaction with cicletanine (CIC)

Smooth muscle cells were cultured from guinea-pig aorta and labelled with⁴⁵Ca⁺⁺ and³²P_i to investigate the possible effect of cicletanine, a new antihypertensive drug, on the release of intracellular Ca⁺⁺ and the metabolism of phosphoinositide induced by histamine.In⁴⁵Ca⁺⁺ labelled cells, histamine increased in a dose-dependent manner the⁴⁵Ca⁺⁺ efflux in the first two minutes. Stimulation of⁴⁵Ca⁺⁺ release was observed with H₁-agonist [2-pyridylethylamine dihydrochloride (2-PEA)] but not with H₂-agonist (dimaprit). In addition, histamine- or 2-PEA- induced⁴⁵Ca⁺⁺ efflux was inhibited by the H₁-antagonists (mepyramine and terfenadine) whereas the H₂-antagonist (cimetidine) was without effect. Similar results were obtained in³²P_i labelled cells; both H₁-agonists (Histamine and 2-PEA) increased the labelling of phosphoinositides. This effect was completely blocked by mepyramine. These results demonstrate that the histamine-induced stimulation of⁴⁵Ca⁺⁺ efflux and phosphoinositide metabolism are mediated through H₁-receptors.In the above systems, cicletanine was as effective as the H₁-antagonist (mepyramine) with an IC₅₀ of 10^–6 M for both⁴⁵Ca⁺⁺ efflux and phosphoinositide metabolism. Blockade of these systems by cicletanine may be part of the mechanism by which this drug produces relaxation of blood vessels and may account for itsin vivo antihypertensive action.     

Evidence that high affinity (3H)clonidine binding cooperates with H2-receptors in the canine coronary smooth muscle membrane

We have investigated the effects of myocardial ischmeia and exogenous histamine and 4-methylhistamine on the regulation of membrane bound ₂-and -adrenoreceptors (ARs) in the canine coronary artery smooth muscle (CAS). The results indicate that exposure of CAS to ischemia and histamine is associated with the stimulation of adenylate cyclase and with a down-regulation of ₂ which is accompanied by the sequestration of ₂ sites into light membrane particles. The increased, number of -AR sites in CAS represents a c-AMP mediated adaptational pathway in compromized CAS.     

Muscarinic M3-receptors mediate cholinergic synergism of mitogenesis in airway smooth muscle

Muscarinic receptor agonists have been considered to act synergistically in combination with growth facors on airway smooth muscle growth. Characterization of the proliferative responses and of the receptor subtype(s) involved has not yet been studied. Therefore, we investigated mitogenesis induced by stimulation of muscarinic receptors, alone and in combination with stimulation by platelet-derived growth factor (PDGF). For this purpose, [(3)H]thymidine-incorporation was measured at different culture stages in bovine tracheal smooth muscle cells. Functional muscarinic M(3)-receptors, as measured by formation of inositol phosphates, were present in unpassaged cells, but were lacking in passage 2 cells. Methacholine (10 microM) by itself was not able to induce a proliferative response in both cell culture stages. However, methacholine interacted synergistically with PDGF in a dose-dependent fashion (0.1-10 microM), but only in cells having functional muscarinic M(3)-receptors. This synergism could be suppressed significantly by the selective M(3)-receptor antagonists DAU 5884 (0.1 microM) and 4-DAMP (10 nM), but not at all by the M(2)-subtype selective antagonist gallamine (10 microM). These results show that methacholine potentiates mitogenesis induced by PDGF solely through stimulation of muscarinic M(3)-receptors in bovine tracheal smooth muscle cells.     

Effect of exercise training volume on arterial contractility and BKCa channel activity in rat thoracic aorta smooth muscle cells

Large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels play a critical role in regulating cellular excitability and vascular tone. Exercise training showed reversible beneficial effects on cardiovascular systems with an improvement of vascular functions. This study investigated the effects of exercise training volume on vascular function and BK_Ca channel activity in thoracic aorta smooth muscle cells (SMCs) in 20 sedentary (SED) and 40 training rats, submitted to a treadmill training protocol (20 m/min, 60 min/day, 12 weeks). Training rats were divided into two groups, exercising 3 days/week (EX1) and 5 days/week (EX2). Since intensity and duration of exercise were identical between training groups, the training volume was higher in EX2 than in EX1. Exercise training not only decreased heart rate, but also attenuated pressor responses induced by angiotensin II or norepinephrine (NE). The maximal vascular contraction induced by 10^?5 M NE was significantly decreased after training. In precontracted thoracic aorta with NE (10^?5 M), activation of the BK_Ca channels by NS1619 significantly decreased the tension. The sensitivity of tissue to NS619 (pD2) was significantly correlated with volume of training (SED < EX1 < EX2). Inside-out patch clamp recording on aortic SMCs showed that exercise training significantly increased the open probability, decreased the mean closed time and increased the mean open time of BK_Ca channels. This effect was more significant in the EX2 group than in the EX1 group. These data suggest that there is a dose effect for exercise training volume for the activation of BK_Ca channels in vascular SMCs, which contributes to improvement of the arterial function in thoracic aortas.     

Effects of chloroquine on smooth muscle contracted with noradrenaline or high-potassium solutions in the rat thoracic aorta.

The effects of chloroquine on the smooth muscle of isolated rat aortic segments were investigated in preparations contracted with either noradrenaline or high-potassium. At rest, chloroquine (up to 10(-4) M) produced no mechanical response, while noradrenaline (10(-6) M) produced a sustained contraction. In the presence of 10(-4) M chloroquine, however, the amplitude of contractions produced by noradrenaline was attenuated by about 70%, with no alteration of the resting tension. In preparations contracted either with noradrenaline or with high-K solutions, chloroquine produced a concentration-dependent relaxation. The tension decreased below resting level as a result of the co-application of these stimulants. The relaxing actions of chloroquine were not altered by methylene blue (an inhibitor of guanylate cyclase), suggesting that the cyclic GMP-related mechanism was not involved. The ratio of the amplitude of chloroquine-induced relaxation was similar in contractions produced by different concentrations of potassium ions, suggesting that chloroquine did not cause relaxation as a result of membrane hyperpolarization. These results suggest that the inhibition of aortic smooth muscle contraction caused by chloroquine is different to that produced by endothelium-derived vasodilating factors. It is possible that the inhibition of aortic smooth muscle contraction by chloroquine involves modulation of the contractile systems and of their regulatory proteins.     

Insulin-induced up-regulation of histamine H1-receptors

Inflammation Research -     

Structure elements of histamine H1- and H2-receptors

Conclusion Naturally, the suggested structure schemes of the histamine receptors do not exhaust all possible complexities of their structure; however, they may be a basis when predicting activity of many newly synthesized compounds.     

Excitation and contraction in bovine tracheal smooth muscle.

1. The smooth muscle layer of the bovine trachea was studied in vitro with the micro-electrode and sucrose-gap techniques. The membrane potential was stable at--47-6 plus or minus 0-98 (S.E. of mean) mV, and there was no spontaneous electrical or mechanical activity. 2. The cell membrane had strong rectifying properties, making it impossible to elicit action potentials by electrical stimulation in normal Krebs Solution. The rectification was abolished by TEA (30 mmol/l), which depolarized the membrane and produced plateau-type action potentials. 3. The spontaneous repetitive action potentials produced by TEA were associated with rhythmic oscillatory contractions of the muscle strips. 4. Histamine caused an increased tone, with superimposed rhythmic fluctuations in tension. The electrical response consisted of depolarization, with rhythmic slow oscillations in potential (slow waves) which were synchronous with the fluctuations in tension. 5. Acetylcholine produced smooth, tonic contractures of tracheal muscle strips, and caused simple depolarization of the membrane. No action potentials were recorded. 6. In calcium-free solutions containing EGTA, the mechanical response to TEA was completely abolished; the response to histamine was greatly reduced; the response to acetylcholine was reduced to a lesser extent. All responses reverted to normal when normal concentrations of extracellular calcium were restored. 7. Lanthanum added to the bathing solution abolished the contraction due to TEA even though the solution contained calcium. It reduced the histamine-induced contraction to 26% of control, and reduced the acetylcholine-induced contraction to 58% of control; extracellular calcium was present throughout. 8. It is suggested that TEA produces contraction by promoting influx of calcium ions into the cytoplasm. Acetylcholine, and to a smaller extent histamine, are less dependent upon the presence of extracellular calcium, and may be capable of releasing calcium sequestered within the cell; acetylcholine appears to be more effective in releasing sequestered calcium.     

Current spread in the smooth muscle of the rabbit aorta

1. The electrical responses of the smooth muscle cells of the rabbit aorta to both extracellular and intracellular stimulation were studied using the partitioned chamber and Wheatstone bridge method.2. No spontaneous electrical activity was recorded when the tissue was soaked in either isotonic or hypertonic Krebs solutions, and strong depolarizing currents also failed to trigger action potentials in either solution.3. The circular muscle of the aorta has cable properties. Mean values in isotonic Krebs solution were 2.1 mm for space constant and 433 msec for time constant.4. The input resistance (mean 12 MOmega) measured with the Wheatstone bridge method was considerably smaller than that calculated from values measured with the partitioned chamber method.5. Electrotonic potentials could be recorded from the smooth muscle of ;injury bundles' although their amplitude was smaller than that from the intact bundle.6. High concentrations of noradrenaline readily induce oscillatory potentials from the aorta in both isotonic and hypertonic Krebs solutions. It was estimated by simultaneous recording with two micro-electrodes that noradrenaline-induced oscillatory potential can conduct in both longitudinal and transverse directions of the smooth muscle.7. These results suggest that the smooth muscle of the aorta behaves like a syncytium or single unit muscle and activation of cells on the inner surface of the media can be induced both by electrotonic current spread and by propagation of oscillatory potentials from the outer cells directly activated by the transmitter.     

Ciliated smooth muscle cells in atherosclerotic lesions of human aorta

One or two rudimentary cilia were observed by electron microscopy in smooth muscle cells (SMCs) of fatty dots and streaks, but not in normal intima of human aorta. Similar organelles are known to occur in many cell types and various species, but to the best of the author's knowledge, have never been found in the SMCs of arterial or other tissues of man in health and disease; recently, they were reported to be present in the SMCs of experimental atherosclerosis in rabbits. The rudimentary cilia observed in this study had a "9 + 0" axoneme (microtubular complex) and differed also in other aspects from the classical cilia. Semiserial sections of SMCs containing a diplosome disclosed that on several occasions both of its constituent centrioles gave rise to rudimentary cilia. SMCs containing cilia or their basal bodies were observed more often in the human than in experimental atherosclerotic lesions. Whereas the function and significance of the rudimentary cilia remain largely unknown, the current theory proposes that a sudden transformation from a mitotic replicative to a nonmitotic structured tissue "diverts" centrioles to the formation of these unusual organelles. It is conceivable that rudimentary cilia could serve as morphological indicators of aborted mitosis in human atherosclerotic lesions.     

Histamine H1- and H2-receptors in coronary arteries of pigs

Histamine exerts its action in smooth muscle via two types of receptors. In coronary arteries of pigs both types of receptors are also involved in responses to histamine. Histamine initiates by an interaction with H₁-receptors a contraction and with H₂-receptors a relaxation. The histamine-induced contraction is reduced by the competitively antagonistic action of mepyramine. Metiamide potentiates dose-dependently the histamine-caused contraction in the absence and presence of mepyramine.The proportion of H₂-receptors in relation to H₁-receptors is calculated.     

Histaminergic (H1) modulation of antinociception in morphinetolerant mice

This study compares the actions of diphenhydramine (a centrally penetrating histamine H₁-antagonist) and acrivastine (a peripherally acting H₁-antagonist) in morphine-tolerant and morphine-naïve mice. Mice receivei morphine twice daily for 5–7 days to induce tolerance. Once tolerant, the mice again received morphine, this time after pre-treatment with diphenhydramine (30 mg/kg s.c.) or acrivastine (10 mg/kg s.c. or 5 g intracerebroventricularly-i.c.v.). Diphenhydramine (s.c.) and acrivastine (i.c.v.) increased the action of morphine in the hot-plate test at 49±1°C. However, acrivastine (s.c.) had no effect. In morphine-naïve mice, diphenhydramine increased the antinociceptive action of morphine (5 mg/kg) as did acrivastine when given intracerebroventricularly but not subcutaneously. Therefore, H₁-antagonists potentiate the antinociceptive action of morphine in both morphine-tolerant and morphine-naïve mice via a central, rather than peripheral, action.     

Altered calcium concentration and rabbit atrial H1-receptors

Rabbit atria were incubated in various concentrations of calcium (0.9, 1.8 and 3.6 mM) and then homogenized and histamine H₁-receptors analysed by radioligand binding using (³H)-pyrilamine. Only atria incubated in 3.6 mM calcium showed an alteration in (³H)-pyrilamine binding. In these tissues theK _d of H₁-receptors for (³H)-pyrilamine was increased suggesting a decrease in the affinity of the H₁-receptor. That this decreased affinity is due to altered calcium concentrations was demonstrated by showing that no change in histamines' chronotropic effects were produced by decreasing the sodium concentration to produce the same calcium/sodium ratio compared to high calcium alone. We conclude that calcium has effects directly on H₁-receptor affinity and appears to produce a decrease in the ability of histamine to bind.     

自体静脉移植后管壁超微结构的变化

目的　探讨自体静脉移植后内皮形态特征和中膜SMC增生特点 ,为静脉搭桥后再狭窄的防治提供资料。方法　兔 18只 ,随机分 6组 ,均作模拟血管搭桥手术 ,即将颈外静脉移植于颈总动脉 ,术后取材 ,光电镜观察。结果　移植后 1～ 4周管壁渐增厚 ,4～ 6周时达最厚 ,随后管壁不再继续增厚。对照侧静脉无微绒毛梭形内皮被移植段具有丰富微绒毛呈不同形态的内皮细胞所替代。移植后 1～ 2周 ,SMC肌丝和致密体减少 ,与合成和分泌有关的细胞器异常丰富 ,SMC从收缩型转变成合成型 ,四周时分泌型细胞器略有减少 ,但细胞间的胶原纤维明显增多。第 6～ 12周 ,SMC又渐从分泌型向收缩型转变。结论　自体静脉移植后 4～ 6周管壁厚度达高峰 ,内皮微绒毛增多。中膜SMC在 1～ 2周转型增殖最明显。提示术后 2周内是药物抑制SMC增殖的良机     

Rectification in the smooth muscle cell membrane of rabbit aorta.

1. The current-voltage relation of the smooth muscle cell membrane of rabbit aorta was determined by the partition method. 2. No anomalous rectification was observed in any of the following solutions: normal Krebs, Na free choline, Na sulphate, and high K-Na free sulphate. 3. Delayed rectification was seen on application of depolarizing current in both normal Krebs solution and Na free choline solution. 4. High concentration of K made the steady-state current-voltage relation almost linear in a voltage range of about 0 to -20mV. This effect, and steady-state cathodal rectification which was seen in physiological solution, could be explained qualitatively by constant field theory without involving channels capable of anomalous rectification. 5. A slow decrease in K conductance, during application of large and long-lasting hyperpolarizing currents, which occurs in skeletal muscle and is attributed to the tubule system, was never observed in the arteries either in Krebs, Na-free choline, or Na sulphate solution.     

Characterisation of histamine-H1 receptor binding sites in bovine, rat and guinea pig thoracic aorta

Radioligand binding studies elucidating the localization of vascular histaminergic-H₁ receptors using³H-mepyramine demonstrate that, in addition to histaminergic-H₁ receptors associated with the vascular smooth muscle membranes of bovine thoracic aorta, these receptor binding sites are also present on the endothelial layer of bovine aorta. The receptor number in the vascular smooth muscle membranes was diminished when the aorta was rubbed of endothelium prior to the membrane preparation (B_max=58.5 vs 53.7 fmol/mg protein). As shown in a further study, vascular smooth muscle histamine receptors are homogeneous (high affinity sites only — K_D=3.11 nM), whereas high and low affinity sites exist in the endothelium (K_D=2.19 nM and 32.0 nM respectively).There are species differences in the binding characteristics between bovine, rat and guinea pig vascular smooth muscle histaminergic-H₁ receptors: bovine and guinea pig vascular histamine receptors are homogeneous (high affinity sites) whereas two affinity sites for³H-mepyramine binding exist in the rat.     

Intima-like smooth muscle cells: developmental link between endothelium and media?

The presence of non-contractile smooth muscle cells within the arterial wall raises questions as to their origin and function. These cells abound within the aortae of murine and porcine neonates, but are also present within the intimal and medial layers of adult arteries. They are largely devoid of smooth muscle-associated proteins and manifest an epithelioid form. Their morphological resemblance to endothelial cells prompted us to explore this potential relationship and to investigate their angiogenic properties in three-dimensional collagen gels. Using well-characterized smooth muscle cell lines, displaying either the intima-like (epithelioid) or media-like (spindle-shaped) morphology, we were able to show that intima-like cells share several features in common with endothelial ones and can transform into a media-like phenotype, whereby they irreversibly lose their characteristic pattern of protein expression. Intima-like, but not media-like, vascular smooth muscle cells are capable of forming capillary tubes, and, in co-cultures, can induce media-like ones to participate in this process. Such capillaries consist of a randomly-organized, mixed population of endothelial cells with intima-like or media-like smooth muscle ones. The functional significance of this diversity in smooth muscle cell type is not well understood, but phenotypic plasticity could conceivably figure as an important adaptive response to changes in the local environment.     

Electrical coupling between smooth muscle and endothelium in arterioles of the guinea-pig small intestine

Equations describing the steady-state passive electrical properties of arterioles have been derived. The arteriole was modelled as having two thin layers of cells (muscle and endothelium) with strong electrical coupling between cells within a layer and variable coupling between the layers. The model indicated that spread of membrane potential changes was highly dependent on the thickness of cells within the layers. The model was also used to identify the optimal experimental strategy for detecting coupling between the two layers, and experiments were carried out on arterioles from the guinea-pig small intestine. Thickness of the endothelial layer was measured using electron microscopy and was found to be around 0.5 microm. Electrical input resistance was measured in intact arterioles and compared to input resistance of arterioles from which the endothelium had been removed. The experiments confirmed that there was a strong electrical coupling between the muscle and endothelium in these vessels.