Induction of guinea pig antibody responses in vitro.

Guinea pig spleen cells cultured together with peritoneal exudate lymphocytes (PEL) were found to generate large numbers of antibody-forming cells (AFC) in vitro in response to hapten-protein antigens. Neither cell type cultured alone yielded appreciable responses. Strain 13 or F1 (Strain 2 X Strain 13) lymphocytes, but not those from strain 2 animals, are able to respond to the genetically controlled antigen, DNP-guinea pig albumin (DNP-GPA). Antisera directed against responder (strain 13) parent Ia antigens selectively blocked the generation of AFC by F1 (strain 2 X strain 13) spleen-PEL mixtures in response to DNP-GPA. Both allogeneic (strain 2) and syngeneic macrophages functioned equally well in presentation of DNP-GPA to strain 13 lymphocytes.     

铃蟾肽介导的豚鼠肠系膜下神经节非胆碱能迟慢兴奋性突触后电位

应用细胞内记录技术,对铃蟾肽(bombesin,BOM)在豚鼠离体肠系膜下神经节(inferior mesenteric ganglion,IMG)非胆碱能兴奋性突触传递中的作用进行了研究。重复电刺激突触前结肠神经,有74．3％(52／70)IMG细胞可诱发迟慢兴奋性突触后电位(ls-EPSP)。在可引出ls-EPSP的细胞中,22％(4／18)细胞同时对BOM和SP敏感。用BOM持续灌流IMG,可明显抑制对BOM敏感细胞的ls-EPSP,对BOM不敏感细胞的ls-EPSP则无影响,且BOM受体与SP受体间无交叉脱敏。BOM受体阻断剂tyr^4[D-phe^12]bombesin能明显可逆性地抑制BOM敏感细胞的ls-EPSP和去极化,但对BOM不敏感细胞则无影响。研究结果提示,BOM可能是介导豚鼠IMG细胞ls-EPSP的一种递质。     

NG-nitro-L-arginine inhibits non-adrenergic, non-cholinergic relaxation in rabbit urethral smooth muscle.

Electrical field stimulation induced a relaxation response in female rabbit urethral smooth muscle strips precontracted with phenylephrine. The relaxation response was inhibited by tetrodotoxin, but not by atropine, propranolol, or hexamethonium. The relaxation response thus results from stimulation of inhibitory non-adrenergic, non-cholinergic nerves. The electrically induced relaxation response was inhibited by an inhibitor of nitric oxide biosynthesis, NG-nitro-L-arginine. This inhibition was overcome by addition of a precursor of nitric oxide, L-arginine. An inhibitor of soluble guanylate cyclase, methylene blue, reduced the relaxation response, and a selective cyclic GMP phosphodiesterase inhibitor, M & B 22948, potentiated the relaxation response. These data indicate that agents which affect the biosynthesis of nitric oxide are associated with the urethral relaxation response evoked by electrical field stimulation, and that cyclic GMP may mediate the relaxation response.     

Effect of bradykinin on airway neural responses in vitro.

We investigated the effects of bradykinin (BK) on airway excitatory nonadrenergic noncholinergic (e-NANC) and cholinergic nerves in vitro. Neural responses were elicited by electrical field stimulation in guinea pig airways in vitro before and after the addition of BK (10(-10)-10(-7) M). Captopril (10(-5) M) and phosphoramidon (10(-6) M) were added to prevent degradation of BK, and all neural responses were measured in the presence of indomethacin (10(-5) M) and propranolol (10(-6) M). BK potentiated e-NANC responses in bronchi in a concentration-dependent manner (10(-10)-10(-7) M) without changing concentration-response curves to exogenously applied substance P (10(-10)-10(-5) M). BK significantly potentiated e-NANC neural constrictor responses by 22 +/- 7% at 10(-8) M (mean +/- SE, n = 5, P < 0.05) and 32 +/- 7% at 10(-7) M (n = 8, P < 0.01), compared with changes in time-matched control tissues (7 +/- 2%, n = 8). The potentiation of e-NANC responses by BK was abolished by pretreatment with a specific B2-receptor antagonist, HOE 140 (10(-7) M). Cholinergic constrictor responses elicited to electrical field stimulation were not affected by the addition of BK (up to 10(-7) M). These results suggest that BK potentiates e-NANC bronchoconstrictor responses prejunctionally via a B2-receptor.     

Adenosine selectively inhibits noncholinergic transmission in guinea pig bronchi

The neuromodulatory action of adenosine and ATP was investigated in isolated guinea pig bronchial strip chain preparations contracted with electrical field stimulation. The tissues were placed in organ baths containing physiological salt solution and stimulated at 8-Hz frequency, 0.5-ms pulse duration, and 30 V (approximately 100 mA) for 5 s. Electrical field stimulation evoked a biphasic contraction of bronchial muscle, consisting of an initial contraction followed by a sustained contraction, which was mediated by intramural cholinergic and noncholinergic nerve stimulations, respectively. Adenosine, at concentrations greater than M, caused a concentration-dependent inhibition in the height of the noncholinergically mediated contraction, accompanied by a very weak inhibition on the cholinergically mediated response. ATP (10(-5) to 3 x 10(-3) M) also produced a similar inhibitory effect on the noncholinergically mediated contraction, but the inhibitory potency was less than that of adenosine. The inhibitory response to adenosine was enhanced by the pretreatment with dipyridamole (2 x 10(-6) M) but antagonized with aminophylline (10(-5) M). Contractions of bronchial muscle evoked by exogenous acetylcholine (2 x 10(-6) M) or substance P (2 x 10(-7) M) were significantly inhibited by the adenosine (3 x 10(-4) M) pretreatment. These data suggest that in isolated guinea pig bronchi adenosine selectively inhibits noncholinergic neurotransmission through prejunctional P1-purinoceptors.     

Repeated antigen challenge induced airway hyperresponsiveness to neurokinin A and vagal non-adrenergic, non-cholinergic (NANC) stimulation in guinea pigs.

The effect of repeated weekly antigen challenges by aerosol on bronchopulmonary responses to ACh, histamine, neurokinin A or atropine-resistant (NANC) component of vagal stimulation, has been studied in guinea pigs. Bronchospastic responses were measured in anaesthetized animals, 7 days after the last challenge with antigen (or vehicle). No difference was observed between control and antigen challenged guinea pigs in their responsiveness to acetylcholine (1-300 mumol kg-1 i.v.) or histamine (1-300 mumol kg-1 i.v.). On the other hand, amplitude of bronchospasm induced by neurokinin A (1-3 mumol kg-1 i.v.) or NANC vagal stimulation (20 Hz, 1 msec, 10 V, trains of 5-20 sec) was significantly increased in guinea pigs previously challenged with antigen, as compared to controls. These results suggest that repetitive antigen exposure in sensitized guinea pigs generates an increase in the responsiveness to exogenously administered or endogenously released tachykinins, at a time when no generalized hyperresponsiveness to other spasmogens could be observed.     

Ultrastructural identification of non-adrenergic,non-cholinergic nerves in the rat anococcygeus muscle

The innervation of the rat anococcygeus muscle has been investigated ultrastructurally following fixation with a modified chromaffin reaction for the demonstration of biogenic amines (Tranzer and Richards, 1976). Three types of nerve profiles were revealed: (1) 60-70% of the profiles are adrenergic; (2) less than 5% of the profiles appear to be cholinergic; (3) up to 40% of the profiles are distinguished by the presence of a characteristically high proportion of electron-opaque, chromaffin-negative vesicles, 85-110 nm in diameter. This third type of profile was not affected by 6-OHDA, and is considered to represent the non-adrenergic, non-cholinergic inhibitory innervation of this tissue. Because of the morphological similarity of this nerve type, apart from the smaller vesicle size, to classical peptidergic nerve endings, they have been termed "small p-type" (sp-type). These results are discussed in relation to a previous report describing only two types of nerve profiles in this tissue (Gillespie and Lüllmann-Rauch, 1974).     

Effect of nedocromil sodium on polymorphonuclear leukocyte plasma membrane

The effect of nedocromil sodium on the plasma membrane fluidity of polymorphonuclear leukocytes (PMNs) was investigated by measuring steady-state fluorescence anisotropy of 1-[4-trimethylammonium-phenyl]-6-phenyl- 1,3,5-hexatriene (TMA-DPH) incorporated in the membrane. Our results show that nedocromil sodium 300 muM significantly decreased membrane fluidity of PMNs. The decrease in membrane fluidity of PMNs induced by fMLP was abolished in the presence of nedocromil sodium. These data suggest that nedocromil sodium interferes with the plasma membranes of PMNs and modulates their activities.     

Effect of nedocromil sodium on aspecific bronchial hyper-reactivity in asthmatic children

The purpose of this study was to evaluate whether nedocromil sodium benefits urban asthmatic children showing seasonal bronchial hyper-reactivity to ultrasonic nebulization of distilled water (UNDW). A prospective, randomized, placebo-controlled, parallel-group, double-blind study was carried out at the outpatient pulmonology service at a tertiary-care teaching hospital. Twelve children living in Milan, who were 7-17 years of age, who were SPT and RAST-negative to perennial allergens, who were suffering from episodic asthma, and showing seasonal bronchial hyper-reactivity to UNDW during winter, participated in this study. All the children received either placebo or nedocromil sodium, 4 mg every 6 h for 6 weeks. Spirometry and UNDW challenge were done at the following times: day-7; day 0; day 1; day 7; day 14; day 28; day 42. No differences were found in the basal spirometric parameters, which were normal in both nedocromil and placebo groups. Bronchial reactivity to UNDW was found to be significantly decreased in the group treated with nedocromtl starting from day 7. It is therefore concluded that nedocromil sodium can reverse bronchial hyper-reactivity caused by seasonal factors such as cold, viral infections and atmospheric pollutants in children suffering from asthma.     

Temperature dependence of synaptic responses in guinea pig hippocampal CA1 neurons in vitro

1. Temperature-dependent properties of synaptic transmission were studied by recording orthodromic responses of the population spike and excitatory postsynaptic potential in CA1 pyramidal neurons of guinea pig hippocampal slices.2. Increasing the temperature of the perfusing medium from 30 to 43°C resulted in a decrease in the amplitude of the population spike (A-PS) and a reduced slope of the excitatory postsynaptic potential (S-EPSP). Bath application of the -aminobutyric acid receptor antagonist, picrotoxin, or a change in the calcium concentration of the perfusate did not affect the A-PS during heating.3. Increasing the strength of the synaptic input to that eliciting a PS with an amplitude 50, 75, or 100% of maximal at 30°C resulted in a significant increase in the A-PS during the middle phase of hyperthermia (35–39°C).4. The long-term potentiation (LTP) induced at either 30 or 37°C showed the same percentage increase in both the amplitude of the population spike and the S-EPSP after delivery of a tetanus (100 Hz, 100 pulses) to CA1 synapses.5. The results of the present study, therefore, indicate that the decrease in CA1 field potential was linearly related to the temperature of the slice preparation, while LTP was induced in these responses during heating from 30 to 37°C.     

Effect of dopamine on amylase secretion from guinea pig pancreatic acinar cells in vitro

Dopamine has been shown to effect pancreatic flow, protein output and amylase secretion in a variety of species. However, there is conflicting evidence regarding the role of dopamine on amylase release in vitro. Specific studies were conducted to evaluate the effect of dopamine and to compare its effects with other substances on basal- and secretagogue-stimulated amylase secretion in a guinea pig dispersed pancreatic acinar cells preparation. Dopamine (10(-6) M) induced a small, but significant (P less than 0.05) increase of amylase secretion. Established secretagogues (10(-6) M) including bombesin, cholecystokinin-octapeptide (CCK-8) and carbachol as anticipated induced significantly larger responses. Other substances tested (10(-6) M) including thyrotropin-releasing hormone (TRH) and muscimol were without effect. Complete dose-response studies (10(-11)-10(-3) M) in the presence of bombesin, CCK-8 and carbachol revealed that dopamine does not affect amylase release in response to these secretagogues. These findings suggest that dopamine is a weak stimulant of amylase secretion in vitro, and that it may therefore play a minor role in regulation of pancreatic enzyme secretion. Several factors including vascular, hormonal and neural have been implicated in regulation of pancreatic exocrine secretion. In particular, autonomic nervous system activity, notably cholinergic, has been shown to affect the secretory status of the pancreatic acinar cell. In addition, several biologically active peptides including bombesin, cholecystokinin (CCK), secretin, vasoactive intestinal peptide (VIP), substance P, gastrin and stimulation of cholinergic (muscarinic) receptors with carbachol have been shown to stimulate pancreatic enzyme secretion both in vivo and in vitro. Certain controversy regarding the role of the sympathetic nervous system in regulation of pancreatic exocrine secretion does exist. For example, several studies with agonists and antagonists of noradrenergic and dopaminergic receptor subtypes suggest a stimulatory effect on pancreatic fluid, electrolyte and enzyme secretion. However, these responses are species-specific and variations inherent to the model have been described. Dopamine administration has been shown to stimulate pancreatic bicarbonate and enzyme secretion in a variety of species including mice, dogs, and man. Radioligand binding studies with 3H-dopamine have revealed the presence of high- and low-affinity dopamine binding sites in dog pancreatic acinar cells. Stimulation of these receptors has been correlated with dose-dependent increases in intracellular cAMP levels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Endomorphins 1 and 2 reduce relaxant non-adrenergic,non-cholinergic neurotransmission in rat gastric fundus

It is now well established that opioids modulate cholinergic excitatory neurotransmission in the gastrointestinal tract. The aim of the present study was to characterize a possible effect of endomorphins on nonadrenergic, noncholinergic (NANC) relaxant neurotransmission in the rat gastric fundus in vitro. The drugs used in the experiments were the endogenous mu-opioid receptors (MORs) endomorphin 1 and 2 and the mu-opioid receptor antagonist CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2). CTAP left the basal tonus and the spontaneous activity of the preparation unchanged. Electrical field stimulation (EFS) under NANC conditions at frequencies ranging from 0.5 to 16 Hz caused a frequency-dependent relaxant response on the 5-hydoxytryptamine (5-HT) (10(-7) M) precontracted smooth-muscle strip. Both endomorphin 1 and endomorphin 2 significantly reduced this relaxation in a concentration-dependent manner. Endomorphin 1 proved to be more potent in reducing the relaxant responses. The endomorphin effects were significantly reversed by the MOR antagonist CTAP. CTAP itself did not influence the EFS-induced relaxation. In summary, these data provide evidence that the endogenous MOR agonists endomorphin 1 and 2 can reduce nonadrenergic, noncholinergic neurotransmission in the rat gastric fundus smooth muscle via a pathway involving MORs. The physiological relevance of these findings remains to be established, since the data presented suggest that the endomorphins act as neuromodulators within NANC relaxant neurotransmission.     

Peptide YY induces nerve-mediated responses in the guinea pig intestine.

The actions of peptide YY (PYY) were studied in longitudinal organ-bath preparations of the guinea pig intestine. PYY induced concentration-dependent (10(-9)-5 x 10(-8) M) relaxations of tissue from the duodenum, jejunum, ileum, and colon. These responses were unaffected by adrenergic blockade and atropine treatment but could be prevented by tetrodotoxin. The pharmacology of PYY actions in segments of the small and large intestine indicated the involvement of intrinsic nonadrenergic, noncholinergic inhibitory neurones in the relaxation response to this peptide. All tissues could be made tachyphylactic to PYY without affecting their ability to respond to the direct acting muscle relaxants ATP or papaverine. Moreover, nicotinic ganglion stimulated relaxations and cholinergic nerve-mediated contractions were also unaffected. These results show applied PYY to have potent neurogenic actions in the guinea pig intestine with some similarities to PYY actions in the rat intestine.     

The conductance of single cardiac sodium channels from guinea pig depends on the intracellular sodium concentration.

Currents through DPI 201-106 modified single sodium channels have been measured in cell-free inside-out patches from guinea-pig ventricular myocytes. Single-channel conductance and reversal potential of the sodium channel have been calculated at different intracellular sodium concentrations [( Na+]i) from microscopic I-V curves, which were obtained by application of linear voltage ramps. The relation between the reversal potential and [Na+]i could be fitted with a modified Goldman-Hodgkin-Katz equation with a relative permeability for K+ over Na+ ions of 0.054. The zero-current conductance of the Na channel as a function of [Na+]i shows a plateau value at low Na concentrations, and increases in a sigmoidal manner at higher concentrations. It is concluded that the Na channel can carry outward currents and that its conductance depends on [Na+]i.     

In vitro effect of calcitonin on guinea pig gallbladder

Calcitonin (CT) is a 32 amino acidic polypeptide hormone which has been found in almost all species and whose effects are mainly concerned with calcium and phosphorous homeostasis. Three preparations are employed for therapeutic uses: salmon (sCT), porcine (pCT) and human CT (hCT). The sCT is the most powerful one and in human volunteers a strong relaxing effect has been shown on gallbladder (GB) basal volume and emptying in response to a meal, intraduodenal instillation of a liquid meal and i.v. cholecystokinin (CCK) infusion. Our study was aimed at investigating if a direct sCT effect could be demonstrated on smooth muscle strips from guinea pig GBs "in vitro" (organ bath). Isometric contractions were measured in response to maximal doses of acetylcholine (ACh: 10(-4) M), KCl (80 mM) and cholecystokinin octapeptide (CCK-OP: 10(-6) M), in absence and in presence of four doses of sCT (1 x 10(-9), 1 x 10(-8), 1 x 10(-7) and 1 x 10(-6) M). sCT did not affect the initial strip basal tone. ACh, CCK-OP and KCl caused, as expected, a powerful contraction of the strips, but no effect was shown when each of the sCT doses was administered before ACh (1.28+ 0.69 SEM without sCT vs 1.28g+ 0.69 with sCT; n = 6) and CCK-OP (1.46g+ 0.19 without sCT vs 1.46g+ 0.19 with sCT; n = 8) or 5 min after the induced KCl contraction. On the basis of these preliminary results, we conclude that no evidence of a direct sCT effect was found on guinea pig GBs when considering either basal smooth muscle tone or isometric contraction in response to ACh, KCl and CCK-OP. Further studies are therefore required to clarify the influence of CT on GB dynamics in vivo and to elucidate its the physiological significance.     

Evidence that adenosine is not involved in the non-adrenergic non-cholinergic relaxation in the rat duodenum.

In rat isolated duodenal segments, adenosine induced, in the presence of atropine and guanethidine, a dose-dependent, long-lasting (about 20 s), tetrodotoxin (TTX)-resistant relaxation both in endoluminal pressure and in isometric tension. Electrical field stimulation (EFS) induced, in the presence of atropine and guanethidine, a TTX-sensitive short-lasting (about 6 s) relaxation followed by a sustained rebound contraction. Theophylline, a P1 receptor antagonist, at the concentration of 100 microM caused a marked inhibition of the adenosine-induced relaxation, while the EFS-induced relaxation was not modified. Our results suggest that adenosine induces relaxation of the rat duodenal smooth muscle acting on P1 receptors localized at muscular level. However, differences in the morphology and in the sensitivity to theophylline between adenosine- and EFS-induced relaxation ruled out adenosine as neurotransmitter of the non-adrenergic, non-cholinergic inhibitory system.     

Effect of stress on the Schultz-Dale reaction in guinea pig aorta.

The smooth muscle of thoracic aorta from guinea pig sensitized with egg albumin (EA) produced an anaphylactic contraction when it was exposed to EA. Experiments were performed to evaluate stress effects on the anaphylactic contraction in guinea pig aortic rings. Two types of stressors were used as immunosuppressor stimuli: physical restraint and shaking of the animals. Both stressors diminished the amplitude of the Schultz-Dale contraction in aortic rings from sensitized guinea pig. The shake stress stimulus interrupted several times during each session induced higher immunosuppression in animals in which the active sensitization and the stress sessions began the same day. Severe restraint stress, prior to active immunization, also suppressed significantly the anaphylactic response. The Schulz-Dale reaction in guinea pig aorta seems to be a valuable technique to study the stress effects on the anaphylactic response.