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Changes in cyclic nucleotide content arc associated with relaxation of lower esophageal sphincter (LES) smooth muscle. Although agents that increase cyclic AMP (cAMP) or cyclic CMP (cGMP) can relax the LES, relaxation produced by activation of enteric neurons is associated with, an increase in intracellular cGMP. To evaluate these changes in another sphincteric area of the gut, ice determined the effects of electrical field stimulation (EFS) (65 V, I ms) and of several relaxant agents that alter cyclic nucleotide content in isolated strips of canine internal anal sphincter. Each strip was contracted with norepinephrine (3 μM). During maximum contraction, tissues were relaxed by EFS or by addition of relaxant agents. EFS produced, a frequency-related relaxation, accompanied by a significant, increase, in cGMP; however, cAMP increased only slightly at the maximum frequency tested (8 Hz). Both EFS-induced relaxation and increased cGMP content were blocked 1 μM tetrodotoxin. Cumulative addition of sodium nitroprusside, forskolin, 8-bromoguanosine 3',5'-cylic monophosphate, or 8-bromoadenosine 3',5'-cyclic monophosphate relaxed the internal anal sphincter in a concentration-dependent manner. Sodium nitroprusside-induced relaxations were accompanied by concentration-dependent increases in cGMP content, whereas forskolin-induced relaxations were accompanied by concentration-dependent increases in cAMP content. In conclusion, both cAMP and cCMP appear to be intracellular mediators of relaxation in the canine internal anal sphincter. In addition, relaxation produced by activation of intrinsic inhibitory neurons is associated with an elevation of intracellular cGMP. Together with our previous findings in the LES, these results suggest that cCMP may be the intracellular mediator of neuronally induced, relaxation of gut sphincteric regions.

Neuronal stimulation of most isolated precontracted gastrointestinal smooth muscle results in relaxation. This study examined the changes in cyclic nucleotide content associated with neuronally induced relaxation in sphincteric and nonsphincteric smooth muscle of the gut. Electrical field stimulation produced a frequency-dependent relaxation of the guinea pig proximal colon (EF50= 1.6 Hz) and taenia coli (EF50= 2.4 HZ), and the canine proximal colon (EF50= 3.1 Hz) and internal anal sphincter (EF50= 3.0 Hz). Changes in cyclic nucleotide content occurred during EFS-induced relaxation. Cyclic AMP levels were significantly increased in the guinea pig poximal colon and taenia coli and in the canine proximal colon. No change in cyclic GMP occurred in these smooth muscles. Conversely, in the canine internal anal sphincter the cyclic GMP content increased significantly with no change in cyclic AMP. These effects in this sphincter are similar to those identified previously in the lower esophageal sphincter of opossum, dog, and man. Tetrodotoxin blocked the EFS-induced relaxation and the associated accumulation of cyclic nucleotides of all tissues. Therefore, EFS-induced relaxation is mediated by the activation of neurons and the release of inhibitory neurotransmitter(s). Also, EFS-induced relaxation is closely associated with increases in smooth muscle cyclic nucleotide content. Most importantly, the change in cyclic nucleotide accumulation that occurs during EFS-induced relaxation is different for sphincteric and nonsphincteric smooth muscle, (i.e., different cyclic nucleotides are generated in sphincteric and nonsphincteric smooth muscle in response to stimulation of nonadrenergic, nonchohnergic, inhibitory enteric neurons).

1. Of the four major phosphodiesterase 4 (PDE4) subtypes, PDE4A, PDE4B and PDE4D are widely expressed in human inflammatory cells, including monocytes and T lymphocytes. We explored the functional role of these subtypes using ten subtype-selective PDE4 inhibitors, each belonging to one of two classes: (i) dual PDE4A/PDE4B inhibitors or (ii) PDE4D inhibitors. 2. These compounds were evaluated for their ability to inhibit antigen-stimulated T-cell proliferation and bacterial lipopolysaccharide (LPS)-stimulated tumour necrosis factor alpha (TNFalpha) release from peripheral blood monocytes. 3. All compounds inhibited T-cell proliferation in a concentration-dependent manner; with IC50 values distributed over an approximately 50 fold range. These compounds also inhibited TNFalpha release concentration-dependently, with a wider ( approximately 1000 fold) range of IC50 values. 4. In both sets of experiments, mean IC50 values were significantly correlated with compound potency against the catalytic activity of recombinant human PDE4A or PDE4B when analysed by either linear regression of log IC50 values or by Spearman's rank-order correlation. The correlation between inhibition of inflammatory cell function and inhibition of recombinant PDE4D catalytic activity was not significant in either analysis. 5. These results suggest that PDE4A and/or PDE4B may play the major role in regulating these two inflammatory cell functions but do not rule out PDE4D as an important mediator of other activities in mononuclear leukocytes and other immune and inflammatory cells. Much more work is needed to establish the functional roles of the PDE4 subtypes across a broader range of cellular functions and cell types.

To study the potential of inflammatory mediators to alter colonic motility, we characterized the response of distal colonic smooth muscle to antigen challenge. Addition of ovalbumin to isolated segments of circular smooth muscle obtained from sensitized guinea pigs produced a biphasic contraction. The initial response consisted of a rapid contraction followed by a late response, which was a more sustained but smaller increase in tone and phasic activity. Interestingly, these two responses could be antagonized differentially. Pretreatment with mepyramine (10 microM) inhibited the initial response, whereas the leukotriene antagonist WY 48252 (10 microM) inhibited the late response. The mast cell stabilizer doxantrazole (0.1 microM) reduced only the late response. Inhibition of cyclooxygenase with meclofenamic acid (1 microM) potentiated both responses, whereas blocking neuronal activity with tetrodotoxin (1 microM) only enhanced the initial response. These data indicate clear differences between the inflammatory mediators important in the initial vs. the late response. The initial response is probably mediated by the release of histamine, with enteric neuronal interactions important in attenuating the magnitude of this response. In contrast, the late response appears to be mediated by the release of peptidyl leukotrienes. In this system, cyclooxygenase products apparently function to decrease the response of the smooth muscle to these mediators. These results suggest that release of mediators during an inflammatory response could profoundly alter colonic motility and that these alterations may be important in the pathophysiological manifestations associated with colonic inflammation.(ABSTRACT TRUNCATED AT 250 WORDS)

Increases in cyclic adenosine monophosphate and cyclic guanosine monophosphate content accompany relaxation of isolated strips of opossum and canine lower oesophageal sphincter muscle. The aim of this investigation was to characterise these responses in isolated muscle from the human lower oesophageal sphincter. Electrical stimulation of enteric neurons produced a frequency dependent relaxation of the human lower oesophageal sphincter that was sensitive to tetrodotoxin. Furthermore, as previously shown in the opossum and canine lower oesophageal sphincter, cyclic guanosine monophosphate content was significantly raised in muscle strips frozen during maximum electrical field stimulation whereas cyclic adenosine monophosphate content was unchanged. In addition, sodium nitroprusside (EC50 = 0.1 microM) produced a concentration dependent relaxation of human lower oesophageal sphincter, significantly increased cyclic guanosine monophosphate content, but did not alter cyclic adenosine monophosphate content. Zaprinast (M&B 22948) and SK&F 94120, selective inhibitors of cyclic guanosine monophosphate and cyclic adenosine monophosphate phosphodiesterases, respectively, both relaxed human lower oesophageal sphincter with a potency similar to that seen in the dog or opossum lower oesophageal sphincter. Finally, the 8-bromo analogues of both cyclic adenosine monophosphate (EC50 = 420 microM) and cyclic guanosine monophosphate (EC50 = 100 microM) relaxed the human lower oesophageal sphincter. These studies suggest that in the human, as well as the canine and opossum lower oesophageal sphincter, increases in cyclic nucleotide content are associated with relaxation and increases in cyclic guanosine monophosphate are associated with the relaxation induced by stimulation of enteric neurons.

Cyclic GMP has been proposed as an intracellular mediator of neuronally-induced relaxation in lower esophageal sphincter (LES) smooth muscle. If cyclic GMP is indeed an intracellular messenger, then agents that activate enteric neurons of the sphincter [e.g. the ganglionic nicotinic receptor agonist dimethylphenylpiperazinium (DMPP)] also should cause a relaxation that is associated with an increase in cyclic GMP content. In isolated smooth muscle strips of canine LES, DMPP produced a rapid relaxation that was accompanied by a significant (P less than 0.05) increase in cyclic GMP content with no change in cyclic AMP content. Pretreatment of tissues with either tetrodotoxin or hexamethonium antagonized both the DMPP-induced relaxation and the associated increase in cyclic GMP. The combination of phentolamine and meclofenamic acid also antagonized both the relaxation and the elevation of cyclic GMP produced by DMPP. Electrical field stimulation (EFS)-induced relaxation and elevation in cyclic GMP was unaltered by meclofenamic acid and phentolamine. In conclusion, DMPP (like neuronal electrical activation) relaxed isolated canine LES through an enteric neuronal inhibitory pathway. The presence of phentolamine and meclofenamic acid did not affect EFS-induced effects, but blocked both the relaxation and the increase in cyclic GMP produced by DMPP, suggesting a more complicated pathway for DMPP in the release of inhibitory transmitter.

1. Of the four major phosphodiesterase 4 (PDE4) subtypes, PDE4A, PDE4B and PDE4D are widely expressed in human inflammatory cells, including monocytes and T lymphocytes. We explored the functional role of these subtypes using ten subtype-selective PDE4 inhibitors, each belonging to one of two classes: (i) dual PDE4A/PDE4B inhibitors or (ii) PDE4D inhibitors. 2. These compounds were evaluated for their ability to inhibit antigen-stimulated T-cell proliferation and bacterial lipopolysaccharide (LPS)-stimulated tumour necrosis factor alpha (TNFalpha) release from peripheral blood monocytes. 3. All compounds inhibited T-cell proliferation in a concentration-dependent manner; with IC50 values distributed over an approximately 50 fold range. These compounds also inhibited TNFalpha release concentration-dependently, with a wider ( approximately 1000 fold) range of IC50 values. 4. In both sets of experiments, mean IC50 values were significantly correlated with compound potency against the catalytic activity of recombinant human PDE4A or PDE4B when analysed by either linear regression of log IC50 values or by Spearman's rank-order correlation. The correlation between inhibition of inflammatory cell function and inhibition of recombinant PDE4D catalytic activity was not significant in either analysis. 5. These results suggest that PDE4A and/or PDE4B may play the major role in regulating these two inflammatory cell functions but do not rule out PDE4D as an important mediator of other activities in mononuclear leukocytes and other immune and inflammatory cells. Much more work is needed to establish the functional roles of the PDE4 subtypes across a broader range of cellular functions and cell types.

The ability of the second generation phosphodiesterase 4 inhibitor SB 207499 (Ariflo), [c-4-cyano-4-(3-cyclopentyloxy-4-methoxyphenyl)-r-l-cyclohexane carboxylic acid], to inhibit inflammatory cytokine production in vivo was evaluated and compared to that of rolipram, a first generation phosphodiesterase 4 inhibitor. To examine human tumor necrosis factor alpha (TNFalpha) production, human monocytes were adoptively transferred into Balb/c mice and challenged with lipopolysaccharide (LPS). In this model, SB 207499 inhibited human TNFalpha production with oral ED50 of 4.9 mg/kg. Similarly, R-rolipram inhibited human TNFalpha production with an ED50 of 5.1 mg/kg, p.o. In contrast to their equipotent activity against TNFalpha production, SB 207499 (ED50 = 2.3 mg/kg, p.o.) was 10-fold less potent than R-rolipram (ED50 = 0.23 mg/kg, p.o.) in reversing reserpine-induced hypothermia, a model of antidepressant activity. In time course studies, SB 207499 (30 mg/kg, p.o.) inhibited TNFalpha production for at least 10 hr; substantial plasma concentrations of SB 207499 were detected over the same interval. The ability of SB 207499 to modulate interleukin-4 production in vivo was assessed in a chronic oxazolone-induced contact sensitivity model in Balb/c mice. In this model, topical administration of SB 207499 (1000 microgram) inhibited intralesional concentrations of interleukin-4 (55%; P.01). The results demonstrate that SB 207499 is a potent inhibitor of inflammatory cytokine production in a variety of settings in vivo. Moreover, although it is as potent as R-rolipram in inhibiting TNFalpha production, it has substantially less central nervous system activity. Thus SB 207499 represents an excellent candidate with which to evaluate the antiinflammatory potential of PDE4 inhibitors.

First-generation phosphodiesterase 4 (PDE4) inhibitors, such as rolipram, inhibit the activation of immune and inflammatory cells. The clinical use of these compounds is limited by gastrointestinal side effects, such as increased acid secretion and nausea. Consequently, the challenge has been to design novel PDE4 inhibitors that maintain the anti-inflammatory actions of rolipram while achieving an improved side effect profile. Among the first of this new class of PDE4 inhibitors specifically designed to have an improved therapeutic index relative to earlier compounds is SB 207499 (Ariflo) [c-4-cyano-4-(3-cyclopentyloxy-4-methoxy-phenyl)-r-1-cyclohexanecarboxyl ic acid]. In this study, we compared the anti-inflammatory and gastric secretogogue activities of SB 207499 with those of rolipram. The cellular models used were (1) histamine release from human basophils, (2) tumor necrosis factor-alpha generation in human monocytes, (3) degranulation of human neutrophils, (4) antigen-driven proliferation and cytokine synthesis from human T cells and (5) acid secretion from isolated rabbit gastric glands. SB 207499 inhibited the activation of a variety of immune and inflammatory cells in a concentration-dependent manner: (1) histamine release in basophils [-log IC25 = 6.6 +/- 0.3 vs. 8.0 for (R)-rolipram], (2) lipopolysacchride-induced TNF-alpha formation in monocytes [-log IC50 = 7.0 +/- 0.1 vs. 7.2 +/- 0.1 for (R)-rolipram], (3) fMLP-induced degranulation in neutrophils [-log IC15 = 7.1 +/- 0.2 vs. 6.4 +/- 0.5 for (R)-rolipram], (4) house dust mite induced-proliferation of peripheral blood mononuclear cells [-log IC40 = 6.5 +/- 0.3 vs. 6.4 +/- 0.3 for (R)-rolipram] and (5) ragweed-induced production of interferon-gamma [-log IC50 = 5.4] and interleukin-5 [-log IC50 = 5.0]. Although SB 207499 inhibits the activation of a variety of immune and inflammatory cells with a potency equal to that of rolipram, it is100-fold less potent than the latter compound as an acid secretagogue [-log EC50 = 6.1 +/- 0.1 vs. 8.3 +/- 0.2 for (R)-rolipram]. Collectively, these data indicate that SB 207499 retains the anti-inflammatory activity of the prototypical PDE4 inhibitor rolipram but is substantially less likely to stimulate gastric acid secretion.

1. The ability of various phosphodiesterase (PDE) inhibitors to reduce the initial and/or late response to ovalbumin (OVA) in isolated strips of guinea-pig colonic smooth muscle from sensitized animals was examined. 2. Both the initial and late responses to OVA (0.05 mg ml-1) were inhibited by the non-selective PDE inhibitor, 3-isobutyl-1-methyl-xanthine (IBMX, EC50 = 26.0 and 6.1 microM, respectively), and the selective inhibitors of low Km cyclic AMP specific PDE (PDE IV), (R)- and (S)-rolipram. The (S)-isomer (EC50 = approximately 1.0 microM for both responses) was about 10 fold less potent than the (R)-isomer (EC50 = approximately 0.1 microM for both responses). 3. Zaprinast, a selective inhibitor of the cyclic GMP-specific PDE (PDE V) inhibited only the late response (EC50 = 1.4 microM). 4. SK&F 94120, a selective inhibitor of the cyclic GMP-inhibited low Km cyclic AMP PDE (PDE III), inhibited the initial response (45.9 +/- 11.9%, P < 0.05) at the highest concentration tested (100 microM), and had no effect on the late response. 5. The results suggest that PDE inhibitors, especially PDE IV inhibitors, can attenuate the contractile response of guinea-pig colon to OVA.

Addition of 5-HT or SKF 103829 (2,3,4,5 tetrahydro-8[methyl-sulfonyl]-1 H-3-benzazepin-7-ol hydrobromide) contracts isolated strips of canine lower esophageal sphincter (LES) circular smooth muscle. 5-HT acts directly on the smooth muscle, since pretreatment with the neurotoxin TTX does not inhibit this contraction. Depletion of extracellular calcium or pretreatment with nifedipine inhibited the contraction to both 5-HT and SKF 103829. Therefore, in this smooth muscle, the contraction produced by both 5-HT and SKF 103829 requires extracellular calcium and is sensitive to inhibition by a voltage-dependent Ca2+ channel antagonist. In addition, with respect to 5-HT, SKF 103829 appeared to act as a partial agonist. Receptor alkylation studies using phenoxybenzamine demonstrated no receptor reserve for the contractile response to 5-HT. Nonsurmountable antagonism of the contraction induced by 5-HT and SKF 103289 was observed with several 5-HT2 antagonists, i.e., methysergide, ketanserin, cyproheptadine, and LY 53857. Using a method established for pseudoirreversible antagonism, the Ki values for these 5-HT2 receptor antagonists were estimated. Results suggested that both 5-HT and SKF 103829 contract the canine LES by interacting at the same receptor site and that this receptor site has characteristics of the 5-HT2 receptor. Finally, neither bulbocapnine, domperidone, nor prazosin significantly alters the response to 5-HT or SKF 103829. Thus, isolated strips of canine LES contain a contractile 5-HT2 receptor, and SKF 103829 behaves as a partial agonist at this site.

Opioid peptides have profound effects on gut motility. To assess their actions on enteric neurons regulating sphincteric smooth muscle, the ability of several opioid agonists to antagonize the neuronally induced relaxation of canine lower esophageal sphincter smooth muscle was examined. Opioid peptides selective for μ (FK 33–824) or δ ([D-Pen 2 ,D-Pen 5 ]enkephalin) receptors produced a concentration dependent inhibition of electrical field stimulation (EFS)-induced relaxation. In contrast, neither κ (ketocycloclazine) or σ (SK & F 10047) opioid agonists were potent inhibitors of EFS-induced relaxation. This inhibition was relatively selective for opioid agonists since BHT 933 (α 2 adrenoceptor agonist) and SK & F 89124 (D 2 dopamine agonist) did not inhibit EFS-induced relaxation. Furthermore, naloxone antagonized the effects of both FK 33–824 and DPDPE. These functional data suggest that opioid receptors are present on sphincteric intrinsic inhibitory neurons and that stimulation of these neuronal receptors can regulate lower esophageal sphincter relaxation.

In isolated strips of opossum lower esophageal sphincter (LES) smooth muscle, elevation of cyclic AMP (cAMP) content is associated with relaxation. Because the activation state of cyclic nucleotide-dependent protein kinases may be a more sensitive measure of functionally important changes in cyclic nucleotide levels, we examined the ability of several pharmacological agents and electrical stimulation of the enteric neurons to activate cAMP dependent-protein kinase (cA-PK) and to relax isolated strips of LES smooth muscle. Addition of either isoproterenol or SKF 94120, a selective inhibitor of the low Km cAMP phosphodiesterase to isolated strips of canine LES produced concentration-dependent increases in the activity ratio of cA-PK and concentration-dependent relaxations of canine LES. In contrast, although both zaprinast (MB 22948), a selective inhibitor of the cyclic GMP selective phosphodiesterase, and sodium nitroprusside relaxed canine LES neither drug significantly increased the activity ratio of cA-PK. Furthermore, electrical stimulation of the enteric neurons produced a frequency-dependent relaxation but did not significantly activate cA-PK. To eliminate the possibility that the rapid metabolism of cAMP prevented us from observing a significant activation of cA-PK during electrical field stimulation, the ability of electrical field stimulation (1.0 Hz) to activate cA-PK was examined in the presence of 0.1 mM 3-isobutyl-1-methylxanthine. At at concentration of 0.1 mM, 3-isobutyl-1-methylxanthine, by itself, significantly increased the activity ratio of cA-PK; however, there was no additional activation produced by electrical field stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Non-Invasive Measurement of Total Serum Bilirubin by Multi-Wavelength Spectral Reflectance by Bili Check (TM) in Newborn Patients † 968

The effect of KC 2450 (racemic 3,5-cis-3-methylamino-2,3,4,5-tetrahydro-1-benzoxepine-5-ol hydrochloride) on lower esophageal sphincter pressure in pentobarbital-anesthetized dogs was determined and compared to the effect of metoclopramide. The ED20 value (i.e. the dose that increased lower esophageal sphincter pressure 20 mm Hg) was 0.72 (0.45-1.04) mg/kg i.v. for KC 2450, significantly different from 2.18 (1.30-3.42) mg/kg i.v. for metoclopramide (P less than 0.01). The superior potency of KC 2450 over metoclopramide also was demonstrated at a dose of 2 mg/kg i.v.; KC 2450 produced an increase in sphincter pressure of 43.2 +/- 4.4 mm Hg and metoclopramide produced an increase in sphincter pressure of only 28.5 +/- 5.4 mm Hg (P less than 0.05). Intraduodenally administered KC 2450 increased lower esophageal sphincter pressure at a threshold dose of 2 mg/kg with 10 mg/kg producing an increase in pressure of 53.2 +/- 9.9 mm Hg. KC 2450-induced increases in sphincter pressure were not affected by bilateral cervical vagotomy or ketanserin, but were eliminated by atropine and reduced by neuronal blockade using tetrodotoxin (TTX). KC 2450 effects also were determined in isolated circular strips of lower esophageal sphincter muscle. KC 2450 produced a concentration-related increase in canine (EC50 = 27 microM) and opossum (EC50 = 199 microM) sphincter muscle strip tension. The KC 2450 concentration-response curve was antagonized by atropine in canine and opossum sphincter muscle strips. Neuronal blockade of canine sphincter muscle with TTX antagonized the KC 2450 concentration-response curve in a non-competitive manner.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of selective alpha adrenoceptor agonists on resting transmembrane potential (Em) and contractile responses of vascular smooth muscle of the canine saphenous vein (CSV) were investigated using microelectrode and isometric tension recording techniques. The Em of the smooth muscle cells of the CSV was -59.5 mV +/- 0.3 (mean +/- S.E., n = 363). The cells were electrically quiescent and did not show spontaneous electrical activity. Norepinephrine (a mixed alpha-1/alpha-2 adrenoceptor agonist), applied in concentrations of 1 X 10(-9) to 1 X 10(-7) M did not depolarize the CSV cell membrane. However, 50% of the maximum contractile response to norepinephrine occurred over this concentration range. At concentrations greater than 1 X 10(-7) M, a dose-dependent contraction and depolarization was observed with norepinephrine. The contractile and depolarization effects of norepinephrine were antagonized by the selective alpha-1 antagonist, prazosin. The selective alpha-1 agonists methoxamine and SKF l-89748 (l-[1,2,3,4-tetrahydro-8-methoxy-5-(methylthio)-2-naphthalenamine] ) caused a dose-dependent depolarization and contraction of the CSV. In contrast, the alpha-2 adrenoceptor agonists, BHT-920 and M-7, could be distinguished from the alpha-1 adrenoceptor agonists by their lack of effect on Em. M-7, at concentrations up to 1 X 10(-6) M, failed to produce a depolarization; however, at 10(-6) M, M-7 produced 80% of its maximum contractile response.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrical field stimulation (EFS) of isolated strips of opossum lower esophageal sphincter (LES) produced a relaxation that was accompanied by an elevation of intracellular cyclic GMP content. In order to compare the time dependence of the EFS-induced relaxation with that of the elevation of cyclic GMP, the ability of EFS to produce relaxation and increase cyclic GMP was measured. The results of these experiments showed that cyclic GMP content increased before the onset of relaxation. Cumulative addition of atriopeptin II, an activator of particulate guanylate cyclase, produced a concentration-dependent relaxation of this tissue and increased cyclic GMP content. In other experiments, zaprinast, an inhibitor of a cyclic GMP selective-phosphodiesterase, produced a concentration-related relaxation of opossum LES and increased cyclic GMP content. However, pretreatment with zaprinast (3 microM) did not potentiate the EFS-induced relaxation or the increase in cyclic GMP content. At this concentration, however, zaprinast increased the basal content of cyclic GMP. Finally, 8-Br-cyclic GMP, a membrane-permeable analog of cyclic GMP, produced a concentration-dependent relaxation of isolated strips of opossum LES. In conclusion, these data extend the initial findings that an elevation in cyclic GMP content is associated with relaxation and suggest that cyclic GMP is a potential intracellular messenger of neurally- and drug-induced relaxation of opossum LES.

The objective of the present experiments was to determine the specific receptor subtype through which dopamine (DA) receptor agonists relax the lower esophageal sphincter in vitro. Opossum lower esophageal sphincter smooth muscle strips were placed in oxygenated Krebs' solution containing propranolol and cocaine. The tissues were placed at a tension that gave maximum relaxation to electrical field stimulation and were then pretreated with phenoxybenzamine. The effects of DA, and the DA receptor agonists epinine and apomorphine were determined. In addition, agonist responses were studied in the presence of the selective DA2 receptor antagonist domperidone, a mixed DA1/DA2 receptor antagonist metoclopramide, and the selective DA1 receptor antagonists bulbocapnine and SK&F 83566. The DA agonists relaxed the smooth muscle strips in the following order of potency: DA greater than epinine greater than apomorphine. Domperidone did not antagonize DA- or apomorphine-induced relaxation. Metoclopramide failed to alter DA-induced relaxation. Bulbocapnine and SK&F 83566 significantly inhibited the relaxation induced by DA. These data indicate that DA-induced lower esophageal sphincter relaxation in vitro is mediated by DA1 receptors.

The dog saphenous vein has postsynaptic subpopulations of both alpha-1 and alpha-2 adrenoceptors which are easily demonstrable using selective agonists and antagonists. Specific alpha-1 (methoxamine and SKF 89748) or mixed alpha-1, alpha-2 (l-norepinephrine and M7) agonists as well as the specific alpha-2 agonist, BHT 920, cause concentration-related contraction of this tissue. However, maximum contractions produced by alpha-2 activation are significantly less than maximum contractions produced by alpha-1 or combined alpha-1, alpha-2 adrenoceptor activation. SKF 89748-induced contractions are competitively inhibited by prazosin (pA2 = 7.74) and rauwolscine (pA2 = 6.63); BHT 920-induced contractions are unaffected by prazosin but inhibited by rauwolscine (pA2 = 8.93). Contractile responses to l-norepinephrine are inhibited by prazosin, rauwolscine or phenoxybenzamine in a manner that suggests that norepinephrine interacts with two subtypes of alpha adrenoceptors in this tissue. These data indicate that the dog saphenous vein strip is a suitable in vitro preparation for study of drug action at both postsynaptic adrenoceptors inasmuch as either subpopulation of alpha adrenoceptor can be studied independently using specific agonists or antagonists.

ACTIVATION OF CYCLIC AMP DEPENDENT PROTEIN KINASB (CA-PK) IS ASSOCIATED WITH RELAXATION OF CANINE LOWER ESOPHAGEAL SPHINCTER (LES) BY ISOPROTERENOL BUT NOT BY ELECTRICAL FIELD STIMULATION (EFS). M.S. Bsmette, M. Graus, T.J. Torphy and H.S. Ormsbee, III. Departmentofmaxgy, Smith Kline 6 French Laboratories, Philadelphia, PA 19101. Relaxation of LES smooth muscle is associated with elevations of either CAMP or cGMP content. Since the biological effects of the cyclic nucleotides are produced through the activation of their respective kinases, we have begun to examine the ability of several drugs and EFS to increase the activity of CA-PK. The gastroesophageal junction was removed from anesthetized dogs. The tissue was opened along the lesser curvature and the mucosa removed. Strips of circular smooth muscle (1.0-1.5 cm hy 0.3 cm) were cut and placed in a” oxygenated Krebs solution at 37“C with a” initial tension of 2 g. Muscle strips were electrically stimulated (4.0 Hz, 1.0 msec, 65V) and only those that gained tqne spontaneously and relaxed to EFS were used. Frequency-response ~“1‘“~s to EFS and cumulative cancentration-response curves to sodium nitroprusside (SNP), and to selective phosphodiesterase inhibitors (M&B 22,948 and SK&F 94120) were constructed in the presence of atropine (luH), phentolamine (IouM), propranolol (luM), and meclofenamic acid (1uM). In experiments examining the action of isoproterenol, propranolol was not added to the buffer. At the conclusion of the concentration-response studies, tissues were flashfrozen for determination of :yclic nucleotide content and CA-PK activity. Cyclic nucleotida’content was measured by RIA. Protein kinase activity was measured by the incorporation of 32Pi into histone (Type IIA) in the presence and absence of CAMP and the activity ratios (-CANP/+CAMP) were determined. I” tissues treated with isoproterenol and SK&F 94120, the activity ratio of CA-Pd was significantly increased. On the other hand EFS, SNP, or M&B 22,94R did not produce a significant change in the activity ratio of CA-PK. This is the first demonstration qf a” activation of PK in smooth muscle of canine LES and supports OUT hypothesis that there exist both CAMP and cGMP mediated pathways of relaxation in LES.