Effects of ovarian steroids upon responses mediated by adrenoceptors in separated layers of the myometrium and in the costo-uterine muscle of the guinea-pig

1 This study describes the effects of ovarian steroid hormones upon the responses to adrenoceptor agonists of isolated myometrium, separated into its longitudinal and circular layers, and of costo-uterine muscle from guinea-pigs. The preparations were field-stimulated at 100 s intervals, and the adrenoceptor agonists phenylephrine and isoprenaline produced enhancement or inhibition of the evoked contractions.

2 Isoprenaline produced propranolol-sensitive inhibitory effects in longitudinal and circular myometrium and costo-uterine muscle preparations from animals from all experimental groups: i.e. from nonsteroid-treated animals (ovariectomized and intact); intact animals treated with either oestrogen or progesterone alone; ovariectomized animals treated with oestrogen; ovariectomized and intact animals treated with progesterone following oestrogen priming; and from animals 1-4 days post-partum. Longitudinal myometrial preparations from progesterone-treated oestrogen-primed and from post-partum animals were most sensitive to this agonist.

3 Phenylephrine produced phentolamine-sensitive excitatory effects in circular myometrial and costo-uterine muscle preparations from animals from all the experimental groups. In contrast, propranolol-sensitive inhibitory responses to phenylephrine occurred in longitudinal myometrial preparations taken from animals treated with progesterone following oestrogen priming, and from post-partum animals. Longitudinal myometrium from animals from the remaining experimental groups exhibited phentolamine-sensitive excitatory responses to phenylephrine.

4 The basis for the selective effect upon the longitudinal myometrium of exposure to progesterone following a period of oestrogen priming, is discussed. The results described are consistent with the possibility that in the longitudinal layer of guinea-pig uterus exposed to progesterone following oestrogen priming there is an increase in the proportion of β-adrenoceptors in this layer. This increase may reduce the likelihood of contractions arising via direct stimulation of α-adrenoceptors in this layer in response to sympathetic activation during pregnancy.

     

The actions of histamine on the separated layers of the guinea-pig uterus: the influence of ovarian steroids

The effects of histamine have been studied on separated circular and longitudinal myometrial layers from the guinea-pig. Virgin guinea-pigs in dioestrus were (1) untreated, (2) treated for 14 days thrice weekly with oestradiol cypionate 20 micrograms/kg s.c., or (3) treated as in (2) but in addition with progesterone 3 mg/animal s.c. daily for 4 days before use. Preparations were field-stimulated (60 V 2 ms 30 Hz for 5 s every 100 s) to elicit regular contractions. Histamine was equipotent in producing enhancement of evoked contractions of circular myometrium from animals in each treatment group (pD2 = 5.03, 5.05 and 4.95 in groups 1, 2 & 3 respectively), and enhanced contractility in all but two longitudinal myometrial preparations. Treatment with oestradiol alone, and with oestradiol and progesterone following oestradiol priming enhanced the magnitude of maximal response and produced a small decrease in the potency of histamine on the longitudinal myometrium. pA2 estimates for mepyramine as an antagonist were similar (8.6) in preparations of longitudinal myometrium from each group of animals. Metiamide (1-10 mumol/l) did not antagonise the effects of histamine in this layer. These results, taken together, indicate that ovarian steroids act selectively on the longitudinal myometrial layer to amplify the action of histamine, but do not alter the histamine receptor subtype which in this layer is exclusively H1.     

β2-ADRENORECEPTOR BINDING SITES IN CIRCULAR AND LONGITUDINAL MYOMETRIAL LAYERS OF THE VIRGIN GUINEA-PIG: THE INFLUENCE OF OVARIAN STEROIDS

• 1 Membranes prepared from both circular and longitudinal muscle layers of the uteri of two groups of virgin adult guinea-pigs were used to study the influence of ovarian steroids upon β-adrenoreceptor binding sites. Animals were (i) treated for 14 days with oestradiol cypionate, beginning on day 9–10 of the oestrous cycle; or (ii) treated as in (i) and in addition, with progesterone for the last four days of oestradiol administration.
• 2 (-)-[¹²⁵I]-iodocyanopindolol ([¹²⁵I]-CYP) was used to determine the numbers and characteristics of β-adrenoreceptor bnding sites in the four membrane preparations. In all cases, binding displayed characteristics of a saturable bimolecular reaction; the estimates of receptor site density (B_Max; 0.26-0.33 pmol g^?1 wet weight) were similar in all four preparations, as were those of binding affinity K_D; 18–31 pmol 1^?1).
• 3 The mean negative logarithms of apparent dissociation constants (pK_D) for the inhibition of specific [¹²⁵I]-CYP binding by ICI118, 551 (β₂-adrenoreceptor selective antagonist) ranged from 8.4 to 8.6; and those for L 643, 717-01J10 (pradrenoreceptor selective antagonist) were 5.7-6.2. Thus the [¹²⁵I]-CYP binding sites in all four membrane preparations displayed the characteristics expected of homogeneous populations of adrenoreceptors of the β₂-subtype. The pK_D values for isoprenaline were also similar in each type of membrane preparation (5.8-6.0).
• 4 It is concluded that the clearcut differences in the contractile responsiveness, to adrenoreceptor agonists, of the circular and longitudinal myometrial preparations from oestrogen-treated guinea-pigs are not due to differences in the numbers, subtype or binding affinities of β-adrenoreceptor binding sites. Moreover, enhancement of the inhibitory potencies of β-adrenoreceptor agonists, in preparations of longitudinal myometrium from oestrogen-primed guinea-pigs treated with progesterone, is not due to an effect of progesterone upon β-adrenoreceptor binding sites.

     

Fenoterol and salbutamol actions on guinea-pig myometrium: effects of ovarian steroids

Responses of isolated preparations of longitudinal myometrium from the guinea-pig to fenoterol, salbutamol and isoprenaline were examined. Virgin adult guinea-pigs were treated (i) with oestradiol cypionate 20 micrograms/kg s.c. thrice weekly for two weeks; or (ii) as in (i) plus progesterone 3 mg/animal for the last four days. Other animals (diestrous; cycle days 6-10) were untreated. The order of potency of the agonists in inhibiting field stimulation-induced contractions of the preparations (isoprenaline greater than or equal to fenoterol greater than salbutamol) indicated interaction with beta 2-adrenoceptors. Oestrogen treatment enhanced the potency of isoprenaline 5-fold. Treatment with oestrogen plus progesterone enhanced the potency of all agonists 10- to 30-fold, and increased the mean maximum response to salbutamol. The mean pKD values for displacement by fenoterol of binding of (-)-[125I]cyanopindolol to membrane preparations from animals in both treatment groups were 5.57-5.92. These results raise the possibility that progesterone may enhance the coupling of beta 2-adrenoceptors to transduction mechanisms in the longitudinal myometrium from oestrogen-primed guinea-pigs.     

α1- AND α2-ADRENOCEPTORS OF THE CIRCULAR MYOMETRIUM FROM THE DIOESTROUS GUINEA-PIG

1. In order to investigate the nature of the alpha-adrenoceptors mediating contraction of circular myometrium from dioestrous guinea-pigs, the effects of several adrenoceptor antagonists upon log concentration curves to noradrenaline and phenylephrine have been examined. 2. In the presence of ICI 118,551 and nisoxetine both phenylephrine and noradrenaline produced concentration-dependent contractures of circular myometrium from virgin dioestrous guinea-pigs. Noradrenaline was the more potent and produced larger maximal contractions. Indomethacin (1 mumol/l) decreased the maximum effects of phenylephrine but not those of noradrenaline. Xylazine produced indomethacin-sensitive contractions which were not dose-related and which never exceeded 40% of those evoked by noradrenaline. Responses to xylazine and to noradrenaline, but not those to phenylephrine, were reduced in a low calcium solution. 3. Prazosin produced competitive antagonism of the effects of phenylephrine upon preparations of circular myometrium from virgin and parous dioestrous animals; pA2 values were both 8.1. Phentolamine also competitively antagonized the effects of phenylephrine (virgin animals, pA2=7.7). 4. Both prazosin and phentolamine antagonized the effects of noradrenaline upon preparations from virgin dioestrous animals, however, Schild plot analysis did not indicate a simple bimolecular interaction between agonist and receptors. In the presence of prazosin the alpha 2-adrenoceptor antagonist idazoxan produced dose-dependent parallel shifts in the positions of the log concentration-response curves to noradrenaline; the pA2 was 7.7. In the presence of idazoxan or of indomethacin prazosin competitively antagonized the effects of noradrenaline; the pA2 values were 8.5 and 8.2 respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Actions of forskolin and isoprenaline on the separated myometrial layers of the guinea-pig uterus: influence of ovarian steroids and pregnancy

Responses to forskolin and isoprenaline of electrically stimulated preparations of longitudinal and circular myometrium were compared. The guinea-pigs used were: untreated (dioestrous), ovarian steroid-treated, pregnant and post-partum. Forskolin inhibited electrically evoked contractions of both myometrial layers obtained from all groups; there was no change in its potency or its maximum effect (90-100% inhibition). The inhibition of contractions of longitudinal myometrium by isoprenaline was enhanced with steroid treatment, attaining a maximum in late pregnancy and immediately post-partum. In circular myometrium, there was no significant change in the potency of isoprenaline on tissues from ovarian steroid-treated animals. However, maximum potency as well as maximum response was observed in late pregnancy. Since the effects of isoprenaline but not of forskolin were modulated, we suggest that ovarian steroids and pregnancy may alter the coupling of beta-adrenoceptors to the adenylate cyclase system.     

On the contractile activity and reactivity of sheep myometrial strips in juvenile,anoestral and gravid periods

• 1.1. Spontaneous contractions of uterine muscle strips from juvenile, anoestral and pregnant sheep were recorded in vitro isometrically. There is a spontaneous contractile activity irrespective of hormonal status of the animals.
• 2.2. Acetylcholine increases the tone and frequency of the phasic contractions of uterine corpus and horn preparations from the three groups of animals.
• 3.3. The adrenaline excitatory effect on the myometrial contractions was mediated by α-adrenoreceptors. The pregnant uterine strips showed strengthening of the inhibitory effect mediated by the β-adrenoreceptors.

     

THE RESPONSE OF THE COSTO-UTERINE MUSCLE OF THE RAT TO ADRENORECEPTOR AGONISTS DURING THE OESTROUS CYCLE: A COMPARISON WITH THE UTERINE HORN

• 1 Adrenoreceptors in the rat costo-uterine muscle were compared with those in the uterine horn. The responses of electrically-stimulated preparations of both tissues to isoprenaline and phenylephrine were investigated in the absence and presence of phentolamine and propranolol to block α- and β-adrenoreceptors respectively. Preparations were taken from rats at known stages of the four-day oestrous cycle.
• 2 Both adrenoreceptor agonists consistently inhibited contractions in both tissues; threshold sensitivity to isoprenaline was similar for both tissues and at all cycle stages. The same held true for phenylephrine, which was only approximately 1/400 as potent as isoprenaline. A generally similar pattern was observed when concentrations producing maximum inhibition were compared; however during proestrus, phenylephrine was more potent in producing maximal responses in uterine horns than in costo-uterine muscles.
• 3 Phentolamine had no effect on inhibitory responses to isoprenaline in either tissue, nor to phenylephrine in costo-uterine muscles, but slightly enhanced the inhibitory action of phenylephrine in uterine horns from proestrous rats. Propranolol blocked the inhibitory effects of both drugs, revealing an excitatory effect of phenylephrine in the uterine horns at proestrus.
• 4 These observations, and estimates of pA₂ values for propranolol against both agonists, indicate that β-adrenoreceptors insensitive to cyclical fluctuations in ovarian hormone levels, are predominant in the uterine horn and may be the exclusive adrenoreceptor present in the costo-uterine muscle. In the uterine horn a small sub-population of excitatory α-adrenoreceptors, the presence of which is masked by β-adrenoreceptors, can be demonstrated during proestrus. These receptors may be dependent upon the presence of the endometrium and/or the circular smooth muscle layer. The costo-uterine muscle, which lacks excitatory α-adrenoreceptors, could serve as a useful model of the longitudinal layer of the myometrium in investigations of drug actions at β-adrenoreceptors.

     

Smooth Muscle Layer-Specific Variations in the Autonomic Innervation of Bovine Myometrium

• 1.To clarify the autonomic innervation regulating longitudinal muscle (LM) and circular muscle (CM) motility in the bovine uterus, functional (nerve stimulation, adrenergic drug responsiveness) and biochemical studies (catecholamine content, radioligand binding) were conducted on parous luteal-phase myometrium.
• 2.Electrical field stimulation (EFS; 60 V, 0.5-msec duration) caused tetrodotoxin (1 μM)-sensitive contractions in a frequency-dependent manner (0.5–20 Hz) in both LM and CM layers.
• 3.The EFS-induced LM contractions were potentiated by propranolol and conspicuously decreased by phentolamine, yohimbine, idazoxan or guanethidine, but were unaffected by prazosin or atropine.
• 4.On the other hand, CM contractions were only slightly decreased by phentolamine, idazoxan, yohimbine and guanethidine, but were insensitive to propranolol, prazosin or atropine.
• 5.The noradrenaline content in LM was about five times higher than that in CM.
• 6.Noradrenaline, adrenaline, clonidine, xylazine, UK14,304 and phenylephrine caused concentration-dependent contractions of both smooth muscle layers.
• 7.Clonidine, UK14,304 and xylazine were more potent contractile agents than noradrenaline and phenylephrine.
• 8.The contractile response to noradrenaline was competitively antagonized by yohimbine, but not by prazosin.
• 9.Binding studies using [³H]-prazosin and [³H]-rauwolscine revealed that the bovine myometrium contained both α₁- and α₂-adrenoceptors, but the α₂-type receptor was dominant in both LM (94% of α-adrenoceptors) and CM (88%) layers.
• 10.The distribution of α-adrenoceptors was muscle layer-specific; that is, the concentration of α₁-receptors in LM was the same as in CM, but the concentration of α₂-receptors in LM was 2.6 times higher than that in CM.
• 11.The results of the present study indicate that there are layer-specific variations in the functional innervation of the parous bovine myometrium (exclusive adrenergic innervation in LM and adrenergic [minor] plus nonadrenergic, noncholinergic innervation [major] in CM), and that α₂-adrenoceptors, which were responsive to the excitatory response of endogenous and exogenous noradrenaline, were dominant in both muscle layers of the bovine myometrium.

     

β-ADRENOCEPTORS IN CIRCULAR AND LONGITUDINAL MYOMETRIAL MEMBRANES AND IN LUNG MEMBRANES FROM DIOESTROUS AND POST-PARTUM GUINEA-PIGS

1. We have examined the binding of (-)[125]-cyanopindolol ((-)[125I]-CYP) to membranes prepared from uterus and lung of dioestrous and post-partum (1-6 days) guinea-pigs. 2. The densities of beta-adrenoceptor binding sites in circular and longitudinal myometrium from dioestrous animals were similar, and approximately one-eight of those in the lung. Ascorbate and ethylenediaminetetraacetic acid in the incubation medium did not affect binding. 3. The numbers and affinities of beta-adrenoceptor binding sites in both myometrial layers and in lung parenchyma from post-partum animals were similar to those in corresponding tissues from dioestrous animals. 4. The distribution of beta-adrenoceptor binding sites in the post-partum uterus was examined using receptor autoradiography. Binding to circular and longitudinal muscle layers and to the endometrium was inhibited by (-)-propranolol (1 mumol/l), by the beta 2-adrenoceptor selective antagonist ICI 118,551 (70 nmol/l), but not by the beta 1-adrenoceptor selective antagonist CGP 20712A (100 nmol/l), indicating that the beta-adrenoceptor present was of the beta 2-subtype. 5. The ability of isoprenaline to compete for (--)[125]-CYP binding sites in uterine membranes from post-partum animals was approximately twice that in corresponding preparations from dioestrous animals. 6. Changes in the numbers or affinity of beta 2-adrenoceptors cannot account for the marked and selective enhancement of the actions of sympathomimetic amines at beta-adrenoceptors previously observed in longitudinal myometrium taken from post-partum guinea-pigs. It is suggested that enhancement of later steps in the chain of events between beta-adrenoceptor occupancy and uterine relaxation, and/or a decrease in the contribution of alpha-adrenoceptors to the net effect of the amine might provide alternative explanations.     

Morphine-tolerant longitudinal muscle strip from guinea-pig ileum

1. Implantation of morphine pellets in guinea-pigs produced a high degree of tolerance and dependence within 3 days.2. The contractions of the longitudinal muscle induced by electrical stimulation of the myenteric plexus-longitudinal muscle preparations obtained from tolerant animals were less depressed by morphine than the contractions evoked in preparations from non-tolerant animals.3. Naloxone did not alter the size of the evoked twitch but antagonized the depressant action of morphine in tolerant and in non-tolerant animals. When given to tolerant guinea-pigs, naloxone caused an increase in intestinal activity in vivo.4. The contractile response of the longitudinal muscle to acetylcholine was the same in preparations obtained from tolerant and non-tolerant animals. Electrically evoked contractions of the myenteric plexus-longitudinal muscle preparations from tolerant animals showed reduced sensitivity to the depressant effects of adrenaline, isoprenaline, and particularly, dopamine.     

A1-and A2-PURINOCEPTORS IN THE GUINEA-PIG UTERUS

1. Radioligand binding and functional studies were undertaken to investigate the P₁-purinoceptors present in the separated myometrial layers and the endometrium of the guinea-pig uterus. 2. In preparations of endometrium-denuded circular myometrium, the A₂-selective agonists (2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamido-adenosine (CGS 21680, 100 μmol/L) and N-ethylcarboxamido adenosine (NECA, 1–10 μmol/L) inhibited contractile responses to phenylephrine. In preparations of endometrium-intact circular myometrium, NECA (10 μmol/L) enhanced responses to phenylephrine. NECA did not modulate the spontaneous contractions of longitudinal myometrium. 3. Homogenate binding studies with circular myometrium, longitudinal myometrium and endometrium revealed saturable high affinity [³H]-NECA binding sites. The mean maximal densities of binding sites (B_max) were 2.08, 14.7 and 15.5 fmol/mg protein, and pK_D (neg. log dissociation constant) values were 9.82, 9.19 and 7.44, respectively. 4. (R-) and (S-) -N⁶-(2-phenylisopropyl)adenosine (R- and S-PIA) both competed for two [³H]-NECA binding sites in preparations of circular myometrium. CGS 21680 competed for two [³H]-NECA binding sites in preparations of endometrium and longitudinal myometrium. All other agonist competition was for one site only. The rank orders of potency of high affinity binding were S-PIA ≥ R-PIA ≥ CGS 21680 (circular myometrium), R-PIA ≥ CGS 21680 ≥ S-PIA (longitudinal myometrium) and CGS 21680 > > S-PIA ≥ R-PIA (endometrium). 5. In preparations of circular myometrium, longitudinal myometrium and endometrium the selective A₁-purinoceptor antagonist, 1,3-dipropyl-8-(2-amino-4-chloro)-phenylxanthine (PACPX), competed for two [³H]-NECA binding sites, the non-selective antagonist 3,7-dimethyl-1-propargylxanthine (DMPX), competed for one site only. 6. NECA increased cyclic adenosine monophosphate (CAMP) levels in preparations of both circular myometrium and endometrium. 7. These results indicate that P₁-purinoceptors of the A₂-subtype mediate the inhibitory effects of adenosine analogues on the phenylephrine-induced contractions of the circular myometrium of the guinea-pig, this effect is modified by the presence of the endometrium. There is no evidence that the [³H]-NECA binding sites of the longitudinal myometrium correlate with functional P₁-purinoceptors in this tissue.     

EFFECTS OF ADRENALINE, ISOPRENALINE AND FORSKOLIN ON PREGNANT HUMAN MYOMETRIAL PREPARATIONS

1. The effects of adrenaline, isoprenaline and forskolin upon the evoked contractions of field-stimulated preparations of human, pregnant, isolated myometrium have been examined. Specimens were obtained at lower segment Caesarean section from patients at 31 (n = 1) and 36-40 (n = 10) weeks of gestation. 2. Adrenaline enhanced the electrically evoked contractions of all preparations studied, indicating that its predominant action on these pregnant myometrial tissues was at alpha- and not beta-adrenoceptors. 3. Isoprenaline in concentrations at and below 10 mumol/l produced inhibitory effects in eight of 11 experiments. In the remaining three experiments, tissues were not responsive to the inhibitory effects of isoprenaline. 4. In all preparations exposed to higher concentrations of isoprenaline (30 or 100 mumol/l), its effects were excitatory. 5. Forskolin produced inhibitory effects on preparations from all uteri, including those from which tissues unresponsive to isoprenaline had been obtained. 6. It is suggested that forskolin in the concentrations which were effective in this study produced its inhibitory effects largely through activation of adenylate cyclase. This implies that the lack of an inhibitory response of some preparations to isoprenaline was not due to reduced activity of the adenylate cyclase system, but that the failure of isoprenaline to produce an inhibitory effect could be due to diminished numbers of beta-adrenoceptors and/or increased numbers of alpha-adrenoceptors, or to a defect in the coupling of the receptors to the adenylate cyclase system. Alternatively, the presence of an endogenous antagonist of the effects of isoprenaline (for example, an eicosanoid), could mask its inhibitory effects. 7. The absence of an inhibitory effect of isoprenaline on some specimens of human gravid myometrium could have clinical implications, in view of the widespread use of beta 2-adrenoceptor agonists as uterine relaxants.     

Investigation of occurrence of tolerance to bronchodilator drugs in chronically pretreated guinea-pigs.

1 The actions of sympathomimetic amines on isolated preparations of atria, trachea and ileum were studied in vitro in guinea-pigs, which had been pretreated for 5 or 12 days, by subcutaneous injection, with adrenaline (5 mug/kg), salbutamol (0.5 mug/kg), salbutamol (0.5 mug/kg), methoxamine (250 mug/kg) or saline (0.9% w/V NaCl solution). 2 In the trachea, a decrease in sensitivity (tolerance) to the relaxant effect of adrenaline was induced by pretreatment, for 12 but not for 5 days, with adrenaline. In these animals, cross-tolerance to isoprenaline or salbutamol was not observed. Tolerance to the relaxant actions of adrenaline isoprenaline or salbutamol was not observed after pretreatment with salbutamol. 3 In the trachea, pretreatment with methoxamine or adrenaline for 12 days did not change the sensitivity to the alpha-adrenoceptor-mediated contractor action of methoxamine. 4 In the atria from those guinea-pigs pretreated with adrenaline or salbutamol, there was no reduced sensitivity to the beta-adrenoceptor agonist actions of adrenaline, isoprenaline or salbutamol. In animals pretreated with methoxamine or adrenaline, there was no observable tolerance or cross tolerance to methoxamine with respect to its alpha-adrenoceptor-mediated positive inotropic action in the atria and no unequivocal evidence of a reduced sensitivity to that action of adrenaline. 5 It was confirmed that the twitch-like contractions of the longitudinal muscle of the electrically stimulated ileum were inhibited by sympatomimetic amines acting on alpha- and beta-adrenoceptors. There was no reduced sensitivity to the inhibitory actions of noradrenaline or isoprenaline on the twitch of ileum isolated from animals pretreated with adrenaline, salbutamol or methoxamine for 5 or 12 days. 6 From our results on these three preparations from the same animals, it is concluded that generalizations regarding changes in sensitivity to sympathomimetic amines following their prolonged administration should not be made in any one species.     

ADRENOCEPTOR AND CHOLINOCEPTOR MEDIATED EFFECTS IN THE ISOLATED URETHRA OF CAT AND GUINEA-PIG

1. Preparations of isolated bladder-urethral junction from cats and guinea-pigs were suspended in Krebs solution (37°C) which was bubbled with carbogen. The urethral lumen was perfused at a very low rate (1–3 ml/h) with Krebs solution. Resistance to flow and changes in longitudinal tension (initial setting 0.5 g) were recorded. In additional experiments, tension changes in urethral circular muscle preparations were registered. 2. The urethral preparations had a basal resistance to flow within the range 5–20 cm H₂O. They had a spontaneous contractile activity that was unaffected by atropine 0·1 μg/ml, phenoxybenzamine 0·1 μg/ml, and tetrodotoxin 0·1–0·5 μg/ml, suggesting it was of myogenic origin. 3. The basal characteristics and the drug-induced changes in the perfused urethras were independent of whether the longitudinal tension was recorded iso-metrically or isotonically, or whether the perfusion was made retrograde or ante-grade. Passive changes in longitudinal tension between 0·5 and 2·0 g did not affect the resistance to flow through the urethra. 4. Adrenaline and noradrenaline, 0·01–1 μg/ml, increased longitudinal tension and resistance to flow in the perfused preparations. The effects, which were sustained and concentration-related, were blocked or reversed into inhibition in the presence of phenoxybenzamine, 0·1 μg/ml. This finding suggests that the stimulatory effects were mediated via a-receptors. 5. Isoprenaline, 0·001–0·005 μg/ml, relaxed and inhibited the activity in the cat urethral smooth muscle and decreased the resistance to flow both under basal conditions and when the urethra was contracted by noradrenaline or acetylcholine. The inhibitory effects were blocked by propranolol, 0·1 μg/ml, suggesting that they were mediated via β-receptors. In guinea-pig preparations, no effect of isoprenaline was observed. 6. Acetylcholine, 0·02–0·5 μg/ml, increased the longitudinal tension and the resistance to flow through the urethras. The latter effect was less pronounced than that produced by noradrenaline, especially in the guinea-pig preparations. Nor-adrenaline was also more effective than acetylcholine in contracting the circular muscle of the urethra. The effect of acetylcholine was blocked by atropine, 0·1 μg/ml, suggesting that the contractions were mediated through muscarinic cholino-ceptors.     

Opioid pathways exert a tonic restraint in the guinea-pig isolated colon: changes after chronic sympathetic denervation

Abstract— We have studied the effects of naloxone on acetylcholine and noradrenaline release in the guinea-pig isolated distal colon, and have assessed the effect of naloxone on electrically-induced contractions of the longitudinal muscle and non-adrenergic, non-cholinergic (NANC) relaxations of the circular muscle coat. Naloxone dose-dependently increased resting and electrically-evoked acetylcholine release and electrically-evoked noradrenaline release. Naloxone was more potent in increasing resting acetylcholine release in colonic specimens obtained after chronic sympathetic denervation. Naloxone (1 μm ) did not affect electrically-induced contractions of the longitudinal muscle, while it enhanced NANC relaxations of the circular muscle. The effects observed with naloxone in the present experiments suggest that opioid pathways exert a tonic restraint on neurotransmission in the guinea-pig colon. After suppression of the adrenergic inhibitory tone, the functional relevance of opioid pathways seems to be increased.     

PHYSIOLOGICAL AND PHARMACOLOGICAL CHARACTERIZATION OF AN INVITRO VAGUS NERVE-TRACHEA PREPARATION FROM GUINEA-PIG

• 1 The trachea with the vagus nerves attached was isolated from guinea-pigs. Contractile responses to nerve stimulation or to drugs were measured as pressure changes in the fluid-filled lumen.
• 2 Electrical stimulation of the vagus nerves caused a prompt increase in the intratracheal pressure with an optimum frequency of stimulation between 20 and 30 Hz. The response to the left vagus was somewhat stronger than the response to the right vagus.
• 3 Carbachol caused a maximum pressure increase which was about twice that achieved by bilateral stimulation of the vagus nerves at 20 Hz. In the presence of physostigmine the two sources of stimuli were equally effective.
• 4 The excitatory response to stimulation of the vagus nerves was completely inhibited by hexamethonium, atropine and terbutaline. This indicates that the excitatory response is mediated via ganglia with end-organ responses mediated exclusively by muscarinic receptors and functionally antagonized by stimulation of β₂-adrenoreceptors.
• 5 The trachea preparation exhibited an intrinsic tone which was reduced by terbutaline and indomethacin but not by atropine or hexamethonium. It is probable that prostaglandins are involved in the generation of intrinsic tone.
• 6 Noradrenaline caused a concentration dependent inhibition of the vagally mediated contractions of the trachea which was antagonized by propranolol and yohimbine. When tracheal tone was induced by carbachol only propranolol was effective thus indicating both pre- and postsynaptic effects of noradrenaline.
• 7 The present study has shown that the isolated vagus nerve-trachea is a stable and useful preparation for the evaluation of drugs acting at various levels of the contractile responses of the trachea.

     

EFFECTS ON BLOOD PRESSURE OF NORADRENALINE AND ISOPRENALINE ADMINISTERED INTO THE THIRD VENTRICLE OF THE BRAIN OF ANAESTHETIZED AND CONSCIOUS CATS

• 1 Noradrenaline administered into the third ventricle of the brain (IIIv) of both conscious and anaesthetized cats induced increases in blood pressure accompanied by small and variable heart rate effects. The pressor responses were reduced after autonomic ganglion blockade indicating their likely central origin.
• 2 Noradrenaline when administered either into a lateral cerebral ventricle (i.c.v.) in conscious and anaesthetized cats or into the cisterna magna (i.c.) in anaesthetized cats induced falls in blood pressure accompanied by bradycardia.
• 3 Pressor responses to IIIv noradrenaline in conscious cats were partly blocked by either propranolol or thymoxamine indicating a possible involvement in the responses of excitatory adrenoreceptors of both α and β types.
• 4 Pressor responses induced by IIIv isoprenaline in conscious cats were blocked by propranolol but not by thymoxamine suggesting the effect is mediated solely via excitatory β-adrenoreceptors.
• 5 In anaesthetized cats prior i.c. administration of noradrenaline reduced the pressor responses induced by IIIv noradrenaline.
• 6 In conscious cats i.v. clonidine reduced pressor responses to IIIv noradrenaline without depressing peripheral vascular noradrenaline sensitivity.
• 7 The results suggest the involvement of excitatory and inhibitory α-adrenoreceptors and of excitatory β-adrenoreceptors in central blood pressure control. It is also concluded that activation of inhibitory α-adrenoreceptors in the hind brain region can suppress the cardiovascular effects of stimulating excitatory α- and β-adrenoreceptors located in the region of the third ventricle.

     

EFFECTS OF PROSTAGLANDIN(PG) E2, D2 I2, 6-KETO-PGF1α AND ARACHIDONIC ACID ON EXCITATORY TRANSMISSION OF CANINE SMALL INTESTINE

• 1 Effects of prostaglandin(PG) E₂, D₂, I₂, 6-keto-PGF_1α and arachidonic acid on the excitatory transmission were examined in the separated longitudinal and circular muscles of canine small intestine in vitro.
• 2 Both muscles contracted in response to electrical field stimulation (1 msec, 20 Hz, 100 pulses, supramaximal voltage: indirect stimulation). The contractions were abolished by treatment with tetrodotoxin (0.3 μM). In the presence of tetrodotoxin, the muscle contracted in response to stimulation using a pulse duration of 20msec (direct stimulation). Sites of action of drugs were determined by the difference in their effects on the responses to indirect and direct stimulation.
• 3 PGE₂ strongly inhibited the contractile response of circular muscle to indirect stimulation whilst slightly inhibiting that to direct stimulation. The data suggest that PGE₂ may act on the transmission prejunctionally and reduce the transmitter release in circular muscle.
• 4 PGD₂ and PGI₂ in high concentrations slightly inhibited the contractile responses of circular muscle to both indirect and direct stimulations, indicating that PGD₂ and PGI₂ slightly inhibited the transmission postjunctionally. 6-keto-PGF_1α did not inhibit the transmission of circular muscle.
• 5 Arachidonic acid inhibited the contractile response of circular muscle to indirect stimulation, and indomethacin attenuated the arachidonic acid-induced inhibition. PGE₂, biosynthetized from arachidonic acid, may act on the transmission of circular muscle.
• 6 In the longitudinal muscle, none of PGE₂, PGD₂, PGI₂ or 6-keto-PGF_1α inhibited the contractile response to indirect stimulation. 7 Arachidonic acid slightly inhibited the contractile response of longitudinal muscle to indirect stimulation. However, indomethacin did not antagonize this effect. 8 The results suggest that the transmission of circular muscle in canine small intestine is prejunctionally regulated by PGE₂, and that of longitudinal muscle is slightly affected by arachidonic acid or by metabolites through lipoxygenase.

     

EFFECTS OF EXPERIMENTALLY INDUCED DIABETES MELLITUS ON PHARMACOLOGICALLY AND ELECTRICALLY ELICITED MYOMETRIAL CONTRACTILITY

• 1 Diabetes is one of the most frequent complications of gestation, affecting approximately 7% of pregnancies. However, little is known about its effects on electrically and pharmacologically stimulated myometrial contractility. The aim of the present study was to investigate the consequences of streptozotocin (STZ)‐induced diabetes on: (i) electrical field stimulation (EFS)‐evoked contraction of isolated uterine rings as a function of gestational age; and (ii) the uterotonic and tocolytic actions of α‐ and β‐adrenoceptor stimulation, respectively. The effects of oxytocin in late pregnancy were also investigated.
• 2 During pregnancy, EFS‐evoked contractions of isolated uterine rings from intact rats declined, whereas isolated uterine rings from diabetic rats exhibited continuously low sensitivity to EFS.
• 3 In non‐pregnant rats, diabetes resulted in increased noradrenaline‐mediated contractility and a decreased relaxation response to terbutaline. At the mRNA level, diabetes enhanced the expression of α_1B‐adrenoceptors in non‐pregnant rats from 14.65 to 18.39 µg/mL (P < 0.05), whereas the expression of α_1D‐adrenoceptors decreased (from 42.87 to 35.67 µg/mL; P < 0.05). During pregnancy, the responses to these sympathomimetics did not differ between diabetic and intact rats.
• 4 In late pregnancy (on Days 15 and 21), oxytocin caused greater maximum contractility of uterine rings from diabetic rats without affecting the EC₅₀. In addition, on Day 15 of pregnancy, the expression of oxytocin receptors in the myometrium of diabetic rats was higher than that in intact rats.
• 5 The results of the present study indicate that experimental diabetes facilitates gestation‐induced denervation and increases myometrial sensitivity to oxytocin in late pregnancy. If similar mechanisms operate in humans, this could contribute to a tendency to premature uterine contractions in diabetes‐complicated pregnancies.