Saul Maayani

Pharmacological studies of human erectile tissue: characteristics of spontaneous contractions and alterations in α‐adrenoceptor responsiveness with age and disease in isolated tissues

1 The pathophysiology of impotence related to vascular smooth muscle dysfunction in the male corpus cavernosum was studied on human isolated erectile tissue (HET). Studies were conducted on 140 sections of HET obtained from 38 male patients undergoing surgery for implantation of penile prostheses to correct underlying erectile dysfunction. 2 Spontaneous myotonic oscillations were characteristic of greater than 90% of all HET preparations at 37°C. These spontaneous oscillations were markedly attenuated by indomethacin, BW755C, nifedipine, removal of extracellular Ca2+, or lower temperatures (≤ 32°C), but were not sensitive to inhibition by atropine, phentolamine or tetrodotoxin. Our data suggest that the oscillations may, at least in part, result from the generation and/or release of a stable cyclo‐oxygenase product and a consequent increase in transmembrane Ca2+ influx. 3 The phenylephrine‐induced contractions in HET may be reliably assayed up to 24 h after surgical removal, without significant alterations in the EC50, maximum response (Emax) or slope index of the steady‐state concentration‐response curve to phenylephrine. 4 The competitive and surmountable nature of the antagonism of phenylephrine‐induced contractions by prazosin and yohimbine allowed calculation of antagonist dissociation constants. The calculated pKb values for prazosin and yohimbine, respectively, were 9.47 ± 0.49 and 5.54 ± 0.22. The rank order of agonist potency in HET was: noradrenaline = phenylephrine ≫ clonidine. These data indicate the presence of a population of membrane receptors that are predominantly of the α1‐adrenoceptor subtype. 5 The entire patient population was stratified on a decennial basis into five age groups, and each age group was subsequently subdivided into diabetic and nondiabetic diagnostic categories. With respect to the steady‐state phenylephrine concentration‐response curves, a Winer two‐factor analysis of variance revealed a significant effect of age on the calculated pEC50 value, as well as a significant age‐diagnosis interaction. A post hoc statistical analysis for unpaired samples yielded significant differences between pEC50 values for diabetic and nondiabetic patients in age groups 41–50 and 61–70 years. In addition, a Winer two‐factor analysis of variance also detected a significant effect of age on the calculated Emax value. 6 In conclusion, our studies demonstrate that spontaneous contractions in HET are likely to be mediated by the generation and release of a stable cyclo‐oxygenase product. Furthermore, the results of both agonist and antagonist studies are consistent with the presence of a homogeneous α1‐adrenoceptor population. Lastly, the responsiveness of isolated HET to phenylephrine was shown to be altered by both age and disease.

Modification of sexual behavior of Long–Evans male rats by drugs acting on the 5-HT1A receptor

Modulation of the sexual behavior of male rats by the anxiolytic buspirone (S-20499) and its analog gepirone were compared to the effects of 8-OH-DPAT (or DPAT, a selective 5-HT1A reference agonist), and BMY-7378 (a selective 5-HT1A partial agonist). Long-Evans rats were used; modulation of copulatory behavior and alteration of penile reflexes were examined. Modulation of copulatory behavior was assessed by three indices: frequency and length of intromission, and latency of ejaculation. DPAT, at doses of 1-8 mg/kg, reduced these three indices in a time dependent manner such that the effects peaked at 45 min and normalized at 90 min. The dose-effect relationship (assessed 45 min after DPAT injection) is bell-shaped with an ED50 approximately 1 mg/kg on the ascending limb of the curve. The effects of buspirone (2 mg/kg) and gepirone (2 mg/kg) on copulatory behavior were indistinguishable from control. BMY-7378 alone and in combination with these other 5-HT1A agonists reduced copulatory behavior, though not statistically significant. Penile reflexes, including number of erections, cups and flips, were inhibited by these agents: DPAT>buspirone>gepirone (inactive at 2 mg/kg). Furthermore, the latency period to erection was at least doubled by DPAT (2 mg/kg). Buspirone and gepirone, however, reduced the latency period to erection. BMY-7378 inhibited penile reflexes when administered alone and even more in combination with DPAT or buspirone. Two butyrophenone analogs, spiperone (a 5-HT1A and dopamine D2 antagonist) and haloperidol (a D2 antagonist), were also tested for their interaction with DPAT. Both of these drugs (at 0.25 mg/kg, 60 min after administration) reduced all indices of penile reflexes and copulation. Furthermore, in combination with DPAT (2 mg/kg, 45 min), the effects were synergistic such that sexual activity came nearly to a standstill. These opposing effects on putatively brain originated copulatory behavior and spinal mediated penile reflexes indicate that the effects of buspirone and DPAT on sexual behavior in the male rat may be possible at different parts of the central nervous system. If a tentative shared target site by DPAT and buspirone is the 5-HT1A receptor, than the same 5-HT receptor sub-type at different locations (brain, raphe nuclei, spinal cord and autonomic ganglia) may modulate rat sexual behavior in opposing ways.

Inhibition of forskolin-stimulated adenylate cyclase activity by 5-HT receptor agonists

We measured the inhibition of forskolin-stimulated adenylate cyclase activity in guinea pig hippocampal membranes by 5-HT, 5-carboxamidotryptamine (CAT) and 8-hydroxy-2-(di-n-propylamino) tetralin (PAT). Low concentrations of these agonists inhibited forskolin-stimulated adenylate cyclase activity in a concentration-dependent and saturable manner. The antagonist spiperone shifted the concentration-response curve to CAT to the right in a parallel manner. The EC50 values of CAT, PAT and 5-HT and the KB of spiperone suggest that this receptor may correspond to the 5-HT1A binding site.

Characterization and radioautography of [3H] LSD binding by rat brain slices in vitro: The effect of 5-hydroxytryptamine

Binding of D-[3H]lysergic acid diethylamide (LSD) to rat coronal brain slices and its blockade by 5-hydroxytryptamine (5-HT) had characteristics similar to those of brain homogenates in respect of KD, kinetics and reversibility of binding. Radioautography was done on slices that had been incubated in 6 nM [3H] LSD and on adjacent slices incubated in the same concentration of tritiated LSD plus 10(-5) M of 5-HT. Choroid plexus showed densest labeling of [3H] LSD. In neuropil, dense labeling occurred within parts of the hippocampal formation except for fields CA2 and CA3 which were sparsely labeled. All layers of the cortex except the posterior cingulate gyrus were labeled by LSD. 5-HT blocked labeling of choroid plexus, hippocampal formation, septum, pons, medulla and parts of cortex but only reduced labeling of most other structures. LSD binding sites may relate to some of its pharmacological effects.

Immunological and respiratory findings in spice-factory workers

Immunological and respiratory findings were studied in a group of 45 female spice-factory workers (mean age: 39 years; mean exposure: 17 years). In addition a group of 45 female control workers matched by sex, age, and smoking habit were also studied. Intradermal skin testing with mixed spice dust allergen demonstrated positive skin reactions in 73.3% of exposed and in 33.3% of control workers (P less than 0.001). Increased IgE serum levels were found in 36.8% of exposed and in 9.7% of the control workers (P less than 0.01). The prevalence of chronic respiratory symptoms was significantly higher in the exposed workers than in the control workers (P less than 0.01). There was, however, no consistent correlation between skin reactivity and chronic respiratory symptoms. There was a high prevalence of acute symptoms during the work shift. These complaints were more frequent in workers with positive skin tests for the symptoms of cough, chest tightness, and irritated and dry throat. Ventilatory capacity was measured by recording maximum expiratory flow-volume (MEFV) curves. There were statistically significant mean reductions during the work shift for all measured lung function parameters in workers with positive skin reactions. In those workers with negative skin reactions only FEF50 and FEF25 reached statistical significance. Aqueous extracts of different spices (chilli pepper, paprika, caraway, coriander leaves, coriander seeds, cinnamon, ginger, onion, curry, and parsley) caused a dose-related contractile response of isolated guinea pig tracheal smooth muscle. These data suggest that immunologic reactions to spices are frequent in spice workers and may be related to acute symptoms and lung function changes, but not to chronic changes. The data further suggest that, in addition to any immunologic response these spices may produce in vivo, they probably also provoke direct irritant reactions in the airways as suggested by in vitro data.

Phencyclidine-induced rotation and hippocampal modulation of nigrostriatal asymmetry

Phencyclidine (PCP) elicited dose-related rotation in naive rats. The effect of PCP was consistent in direction and magnitude from one week to the next but was dissimilar to the rotatory effects of dopaminergic (D-amphetamine, apomorphine) or anticholinergic (scopolamine) drugs. Study of the effects of PCP on regional brain uptake of labeled 2-deoxy-D-glucose suggested that PCP-induced rotation is at least in part mediated by an action in the hippocampus. PCP elicited ipsilateral rotation following unilateral hippocampal lesions whereas such lesions did not alter the direction of either nocturnal or D-amphetamine-induced rotation. PCP appears to activate a hippocampal mechanism that normally only modulates the intensity of rotation.

Nitric Oxide Synthase in Acute Alteration of Nitric Oxide Levels after Subarachnoid Hemorrhage

OBJECTIVE:Subarachnoid hemorrhage (SAH) is associated with acute decreases and subsequent recovery of cerebral nitric oxide (NO) levels, but the mechanisms of these alterations are not known. In this study, we measured NO synthase (NOS) protein and kinetics to determine its involvement in the alterations of cerebral NO levels after SAH. METHODS:The endovascular rat model of SAH was used. The number of NOS-1 (neuronal) and NOS-2 (inducible)-positive cells (0–96 h) was determined by counting immunoreactive cells in 8-μm cryostat sections. The tissue content of active NOS and its kinetic parameters were studied with an enzymatic l-citrulline assay. RESULTS:The number of NOS-1-positive cells increased between 1 and 3 hours after SAH, decreased to and below control values at 6 and 72 hours after SAH, and increased to control values 96 hours after SAH. The number of NOS-2-positive cells increased 1 hour after SAH, decreased to control values at 24 hours, and increased above control values 96 hours after SAH. The Michaelis-Menten kinetic parameters (Vmax, Km, slope) of NOS remained unchanged at 10 and 90 minutes after SAH. CONCLUSION:NOS-1 and -2 proteins undergo a triphasic alteration after SAH, whereas the amount of active NOS and its kinetic parameters remain unchanged during the first 90 minutes after SAH. Depletion of NOS is not involved in the acute alterations of cerebral NO levels after SAH.

Immunological and respiratory changes in soy-bean workers

SummaryImmunological and respiratory findings were studied in a group of 19 male soybean workers. Twenty control workers also participated in the immunological studies. All soybean workers had positive immediate skin reactions to soybean extract, as did 19/20 control workers. Similarly, 18/19 soy workers reacted to soy antigen prepared after separation from oil, but only 3/19 to soy lecithin antigen and 1 to soy oil antigen. A majority of soy workers (13/19) reacted to house dust. Only 3/19 soy workers had increased levels of soy-specific IgE. The prevalence of chronic respiratory symptoms was higher in exposed than in control workers, being significantly different for dyspnea (P < 0.01). Workers with increased specific serum IgE or positive skin tests to house dust did not have any more symptoms than workers with negative tests. Ventilatory function was significantly worse in soybean workers than expected. Nevertheless, workers with positive skin or serological tests to house dust had across-shift changes similar to those with negative tests. These data suggest that skin and airway responses to soybean components (particularly the non-lipid ones) are very frequent among soybean workers. In the current study specific (soy) and non-specific (house dust) skin tests and immunoglobulins did not allow us to identify the workers at risk of developing symptoms or lung function abnormalities. This suggests that in addition to any atopic mechanisms, the irritant effect of soy dust may play a role in this occupational airway disease.

Activation of the 2A –adrenoceptor mediates deceleration of the deaggregation component of the response to ADP or 5-HT in human platelets in vitro

Platelet aggregation requires the concomitant activation of at least one G(i)- and one G(q)-coupled receptor. Epinephrine (EPI) amplifies the response elicited by a number of agonists for platelet aggregation. This study tested the hypothesis that platelet alpha(2A)-adrenoceptor activation causes deceleration of the deaggregation component of the platelet aggregation response when activated concomitantly with the G(q)-coupled adenosine diphosphate (ADP) P2Y(1) or 5-hydroxytryptamine(2A) receptor. The time course of the aggregation response of human platelet-rich plasma following activation of combinations of two or three receptors was assessed by turbidometry using lepirudin anticoagulation. Simultaneous activation of specific two- and three-receptor combinations was achieved using selective antagonists for the P2Y(12) and P2Y(1) receptor subtypes. Steady-state and kinetic parameters, obtained using a four-compartment kinetic model, were used to assess the effects on the net aggregation response. Graded alpha(2A)-adrenoceptor activation was associated with a concentration-dependent decrease of the rate constant of deaggregation. Activation of both ADP receptor subtypes and the alpha(2A)-adrenoceptor produced a concentration-dependent, mutual amplification of the aggregation response. In addition, when three receptors were simultaneously activated, mutual amplification of the aggregation response was observed at physiologically relevant concentrations of epinephrine or norepinephrine (NE) and ADP. We propose that similar to the P2Y(12) receptor, activation of the alpha(2A)-adrenoceptor decelerates the deaggregation component shifting the balance toward increased net aggregation. The effects of EPI and NE on the aggregation response may contribute to the mechanism of increased thrombotic risk present in certain pathophysiological and disease states.Platelet aggregation requires the concomitant activation of at least one G i - and one G q -coupled receptor. Epinephrine (EPI) amplifies the response elicited by a number of agonists for platelet aggregation. This study tested the hypothesis that platelet f 2A -adrenoceptor activation causes deceleration of the deaggregation component of the platelet aggregation response when activated concomitantly with the G q -coupled adenosine diphosphate (ADP) P2Y 1 or 5-hydroxytryptamine 2A receptor. The time course of the aggregation response of human platelet-rich plasma following activation of combinations of two or three receptors was assessed by turbidometry using lepirudin anticoagulation. Simultaneous activation of specific two- and three-receptor combinations was achieved using selective antagonists for the P2Y 12 and P2Y 1 receptor subtypes. Steady-state and kinetic parameters, obtained using a four-compartment kinetic model, were used to assess the effects on the net aggregation response. Graded f 2A -adrenoceptor activation was associated with a concentration-dependent decrease of the rate constant of deaggregation. Activation of both ADP receptor subtypes and the f 2A -adrenoceptor produced a concentration-dependent, mutual amplification of the aggregation response. In addition, when three receptors were simultaneously activated, mutual amplification of the aggregation response was observed at physiologically relevant concentrations of epinephrine or norepinephrine (NE) and ADP. We propose that similar to the P2Y 12 receptor, activation of the f 2A -adrenoceptor decelerates the deaggregation component shifting the balance toward increased net aggregation. The effects of EPI and NE on the aggregation response may contribute to the mechanism of increased thrombotic risk present in certain pathophysiological and disease states.

Immunological findings in hemp workers

Immunological status and its relation to respiratory findings were studied in 42 female textile workers occupationally exposed to hemp dust and in 49 female control workers. Skin prick tests with hemp or flax dust extracts from different parts of the mill in hemp workers demonstrated the following frequencies of positive tests to antigens: a mixture of hemp and flax extracts (64%), followed by flax extracts (48%), hemp from combing machines (41%), hemp from carding machines (38%), hemp from spinning and weaving machines (33%), and hemp from softening machines (20%). The prevalence of positive skin tests to hemp or flax allergens in control workers was consistently lower, ranging from 21 to 5%. Increased total serum IgE was recorded in 35.7% of hemp workers compared to only 5.0% of control workers (P < 0.05). Hemp workers with positive skin tests had significantly higher prevalences of chronic respiratory symptoms than those with negative skin tests. There were, however, no differences for acute symptoms between workers with positive and negative skin tests. Across-shift changes and baseline lung function were not different when compared by immunologic status. We showed additionally that a water-soluble extract of hemp dust causes a dose-related contraction of nonsensitized guinea pig tracheal smooth muscle when studied in vitro. Our results suggest that frequent immunologic abnormalities can be documented in hemp workers but, with the exception of chronic respiratory symptoms, in general, these do not correlate with respiratory findings.