Enhancing GABAergic Transmission Improves Locomotion in a Caenorhabditis elegans Model of Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is a neuromuscular disease characterized by degeneration of spinal motor neurons resulting in variable degrees of muscular wasting and weakness. It is caused by a loss-of-function mutation in the survival motor neuron (SMN1) gene. Caenorhabditis elegans mutants lacking SMN recapitulate several aspects of the disease including impaired movement and shorted life span. We examined whether genes previously implicated in life span extension conferred benefits to C. elegans lacking SMN. We find that reducing daf-2/insulin receptor signaling activity promotes survival and improves locomotor behavior in this C. elegans model of SMA. The locomotor dysfunction in C. elegans lacking SMN correlated with structural and functional abnormalities in GABAergic neuromuscular junctions (NMJs). Moreover, we demonstrated that reduction in daf-2 signaling reversed these abnormalities. Remarkably, enhancing GABAergic neurotransmission alone was able to correct the locomotor dysfunction. Our work indicated that an imbalance of excitatory/inhibitory activity within motor circuits and underlies motor system dysfunction in this SMA model. Interventions aimed at restoring the balance of excitatory/inhibitory activity in motor circuits could be of benefit to individuals with SMA.

Improving vagal activity ameliorates cardiac fibrosis induced by angiotensin II: in vivo and in vitro.

Cardiac remodeling is characterized by overactivity of the renin-angiotensin system (RAS) and withdrawal of vagal activity. We hypothesized that improving vagal activity could attenuate cardiac fibrosis induced by angiotensin II (Ang II) in vivo and in vitro. Rats were subjected to abdominal aorta constriction (AAC) with or without pyridostigmine (PYR) (31 mg/kg/d). After 8 weeks, PYR significantly decreased Ang II level, AT1 protein expression, and collagen deposition in cardiac tissue and improved heart rate variability, baroreflex sensitivity and cardiac function, which were abolished by atropine. In vitro, treatment of cardiac fibroblasts (CFs) with Ang II (10(-7) M) increased cell proliferation, migration, transformation, and secretory properties, which were significantly diminished by acetylcholine (ACh, 10(-6) M). Subsequently, Ang II significantly increased collagen type I expression as well as metalloproteinase (MMP)-2 expression and activity. Transforming growth factor (TGF)-ß1 expression and Smad3 phosphorylation presented a similar trend. Notably, the knockdown of the acetylcholine M2 receptor by siRNA could abolish ACh anti-fibrotic action. These data implicated cholinesterase inhibitor can increase vagal activity and reduce local Ang II level, and ACh inhibit Ang II pro-fibrotic effects. Our findings suggested that the parasympathetic nervous system can serve as a promising target for cardiac remodeling treatment.

Acetylcholinesterase Inhibition Attenuates the Development of Hypertension and Inflammation in Spontaneously Hypertensive Rats.

BACKGROUND: It is hypothesized that chronic increase of availability of acetylcholine, resulting from the effect of antiacetylcholinesterases, may prevent autonomic imbalance and reduce inflammation yielding benefic effects for cardiovascular disorders in hypertension. The effect of long-term administration of antiacetylcholinesterase agents with central and/or peripheral action, i.e., donepezil and pyridostigmine, were investigated on arterial pressure (AP), sympathovagal balance, plasma cytokine levels, and cardiac remodeling in spontaneously hypertensive rats (SHR). METHODS: Chronic treatment with donepezil or pyridostigmine started before the onset of hypertension. AP was measured by plethysmography every 4 weeks. At the end of 16 weeks of treatment, methylatropine was used to evaluate the cardiac vagal tone; AP and pulse interval (PI) variability were also evaluated followed by plasma and heart collection for analysis. RESULTS: Pyridostigmine, which does not cross the blood-brain barrier, increased cardiac vagal tone, and reduced cardiomyocyte diameter and collagen density, but did not affect the AP and plasma cytokine levels. Donepezil, which crosses the blood-brain barrier, attenuated the development of hypertension, increased cardiac vagal tone, and improved AP and PI variability. Likewise, donepezil reduced the plasma levels of tumor necrosis factor-α, interleukin 6, and interferon γ, besides reducing cardiomyocyte diameter and collagen density. CONCLUSIONS: Donepezil attenuated the development of hypertension in SHR probably involving antiinflammatory effects, indicating that acetylcholinesterase inhibition yields benefic effects for antihypertensive therapy.

In vitro kinetic interactions of pyridostigmine, physostigmine and soman with erythrocyte and muscle acetylcholinesterase from different species.

The low effectiveness of atropine and oxime treatment in soman poisoning may be enhanced by carbamates pre-treatment. For ethical reasons medical countermeasures can only be tested in animal models despite the fact of substantial species differences. With this kinetic in vitro study the interactions between pyridostigmine, physostigmine and soman with human, Rhesus monkey, swine and guinea pig erythrocyte AChE were investigated. In addition, the effect of the carbamates on the residual activity and enzyme recovery after soman inhibition was examined with erythrocyte and intercostal muscle AChE from these species with a dynamic in vitro model with real-time determination of AChE activity. Only small to moderate species differences of the inhibition and decarbamylation kinetics were recorded. It was possible to show that with erythrocyte and muscle AChE a similar level of protection by carbamates and reactivation after discontinuation of the carbamates and soman could be observed. Thus, these data indicate that carbamate pre-treatment is expected to protect a critical level of muscle AChE and confirm the presumption that erythrocyte AChE may serve as a surrogate for synaptic AChE. Hence, these and previous data fortify the notion that erythrocyte AChE is a proper tool for in vitro kinetic studies as well as for therapeutic monitoring in experimental and clinical studies.

Protection by pyridostigmine bromide of marmoset hemi-diaphragm acetylcholinesterase activity after soman exposure.

Pyridostigmine bromide (PB) was approved by the U.S. Food and Drug Administration (FDA) in 2003 as a pretreatment in humans against the lethal effects of the irreversible nerve agent soman (GD). Organophosphate (OP) chemical warfare agents such as GD exert their toxic effects by inhibiting acetylcholinesterase (AChE) from terminating the action of acetylcholine at postsynaptic sites in cholinergic nerve terminals (including crucial peripheral muscle such as diaphragm). As part of the post-marketing approval of PB, the FDA required (under 21CFR314, the "two animal rule") the study of a non-human primate model (the common marmoset Callithrix jacchus jacchus) to demonstrate increased survival against lethal GD poisoning, and protection of physiological hemi-diaphragm function after PB pretreatment and subsequent GD exposure. Marmosets (male and female) were placed in the following experimental groups: (i) control (saline pretreatment only), (ii) low dose PB (12.5 microg/kg), or (iii) high dose (39.5 microg/kg) PB. Thirty minutes after the PB dose, animals were challenged with either saline (control) or soman (GD, 45 microg/kg), followed 1 min later by atropine (2mg/kg) and 2-PAM (25mg/kg). After a further 16 min, animals were euthanized and the complete diaphragm removed; the right hemi-diaphragm was frozen immediately at -80 degrees C, and the left hemi-diaphragm was placed in a tissue bath for 4h (to allow for decarbamylation to occur), then frozen. AChE activities were determined using the automated WRAIR cholinesterase assay. Blood samples were collected for AChE activities prior to PB, before GD challenge, and after sacrifice. RBC-AChE was inhibited by approximately 18% and 50% at the low and high doses of PB, respectively, compared to control (baseline) activity. In the absence of PB pretreatment, the inhibition of RBC-AChE by GD was 98%. The recovery of hemi-diaphragm AChE activity after the 4h wash period (decarbamylation) was approximately 8% and 17%, at the low and high PB doses, respectively, compared with the baseline (control) AChE activity prior to PB pretreatment or soman exposure. The results suggest that PB pretreatment protects a critical fraction of AChE activity in the marmoset diaphragm, which is sufficient to allow the animal to breathe despite exposure to a dose of soman that is lethal in unprotected animals.

Acute electrophysiologic consequences of pyridostigmine inhibition of cholinesterase in humans

The cardiovascular electrophysiologic basis for the action of pyridostigmine, an acetylcholinesterase inhibitor, has not been investigated. The objective of the present study was to determine the cardiac electrophysiologic effects of a single dose of pyridostigmine bromide in an open-label, quasi-experimental protocol. Fifteen patients who had been indicated for diagnostic cardiac electrophysiologic study underwent two studies just before and 90-120 min after the oral administration of pyridostigmine (45 mg). Pyridostigmine was well tolerated by all patients. Wenckebach nodal anterograde atrioventricular point and basic cycle were not altered by pyridostigmine. Sinus recovery time (ms) was shorter during a 500-ms cycle stimulation (pre: 326 ± 45 vs post: 235 ± 47; P = 0.003) but not during 400-ms (pre: 275 ± 28 vs post: 248 ± 32; P = 0.490) or 600-ms (pre: 252 ± 42 vs post: 179 ± 26; P = 0.080) cycle stimulation. Pyridostigmine increased the ventricular refractory period (ms) during the 400-ms cycle stimulation (pre: 238 ± 7 vs post: 245 ± 9; P = 0.028) but not during the 500-ms (pre: 248 ± 7 vs post: 253 ± 9; P = 0.150) or 600-ms (pre: 254 ± 8 vs post: 259 ± 8; P = 0.255) cycle stimulation. We conclude that pyridostigmine did not produce conduction disturbances and, indeed, increased the ventricular refractory period at higher heart rates. While the effect explains previous results showing the anti-arrhythmic action of pyridostigmine, the clinical impact on long-term outcomes requires further investigation.

Acute electrophysiologic consequences of pyridostigmine inhibition of cholinesterase in humans.

The cardiovascular electrophysiologic basis for the action of pyridostigmine, an acetylcholinesterase inhibitor, has not been investigated. The objective of the present study was to determine the cardiac electrophysiologic effects of a single dose of pyridostigmine bromide in an open-label, quasi-experimental protocol. Fifteen patients who had been indicated for diagnostic cardiac electrophysiologic study underwent two studies just before and 90-120 min after the oral administration of pyridostigmine (45 mg). Pyridostigmine was well tolerated by all patients. Wenckebach nodal anterograde atrioventricular point and basic cycle were not altered by pyridostigmine. Sinus recovery time (ms) was shorter during a 500-ms cycle stimulation (pre: 326 +/- 45 vs post: 235 +/- 47; P = 0.003) but not during 400-ms (pre: 275 +/- 28 vs post: 248 +/- 32; P = 0.490) or 600-ms (pre: 252 +/- 42 vs post: 179 +/- 26; P = 0.080) cycle stimulation. Pyridostigmine increased the ventricular refractory period (ms) during the 400-ms cycle stimulation (pre: 238 +/- 7 vs post: 245 +/- 9; P = 0.028) but not during the 500-ms (pre: 248 +/- 7 vs post: 253 +/- 9; P = 0.150) or 600-ms (pre: 254 +/- 8 vs post: 259 +/- 8; P = 0.255) cycle stimulation. We conclude that pyridostigmine did not produce conduction disturbances and, indeed, increased the ventricular refractory period at higher heart rates. While the effect explains previous results showing the anti-arrhythmic action of pyridostigmine, the clinical impact on long-term outcomes requires further investigation.

Miastenia gravis en el anciano: experiencia de un hospital comarcal en una patología emergente / Myasthenia gravis in the elderly: a county hospital experience of an emerging disease

Objetivo: evidenciar el significativo incremento de la incidencia de miastenia gravis (MG) en la población anciana, así como conocer las particularidades del manejo de esta enfermedad en esos pacientes. Metodología: análisis retrospectivo de los casos de MG en pacientes mayores de 65 años atendidos en el hospital comarcal de la Merced de Osuna, Sevilla, durante los años 1995 a 2003.Resultados: obtuvimos una serie de 9 pacientes (5 varones y 4 mujeres) con una edad media de 77,6 (ñ 8,6) años y edad media de inicio de 68,1 (ñ 13,8) años. El tiempo medio de evolución era de 9,3 (ñ 11) años. Ocho pacientes (88,8 por ciento) presentaron MG de inicio tardío. Clínicamente 7 pacientes (77,7 por ciento) presentaron la forma ocular (grupo I) y 2 la generalizada (22,3 por ciento), uno leve (grupo IIa) y otro moderada (grupo IIb). Los síntomas oculares fueron las manifestaciones clínicas iniciales preponderantes (88,8 por ciento). Sólo uno de los pacientes presentó timoma. Los anticuerpos antirreceptores de acetilcolina (Ac-AchR) fueron positivos en todos los casos. Todos los pacientes recibieron tratamiento con piridostigmina, y en 5 pacientes (55,5 por ciento) se usaron además esteroides. No se produjo ninguna muerte, aunque en 3 enfermos (33,3 por ciento) los resultados del tratamiento fueron peores. Conclusiones: es necesario cambiar el concepto clásico de MG como patología del adulto joven y considerarla, como refleja nuestro estudio, como una patología emergente en la población anciana. Creemos que la MG es una enfermedad que debemos conocer, dado que la detección y el tratamiento precoces consiguen generalmente una mejoría física y funcional del paciente mayor (AU)

Acetylcholinesterase inhibition: a novel approach in the treatment of neurogenic orthostatic hypotension.

BACKGROUND: Pharmacological treatment of orthostatic hypotension is often limited because of troublesome supine hypertension. OBJECTIVE: To investigate a novel approach to treatment using acetylcholinesterase inhibition, based on the theory that enhanced sympathetic ganglion transmission increases systemic resistance in proportion to orthostatic needs. DESIGN: Prospective open label single dose trial. MATERIAL: 15 patients with neurogenic orthostatic hypotension caused by: multiple system atrophy (n = 7), Parkinson's disease (n = 3), diabetic neuropathy (n = 1), amyloid neuropathy (n = 1), and idiopathic autonomic neuropathy (n = 3). METHODS: Heart rate, blood pressure, peripheral resistance index (PRI), cardiac index, stroke index, and end diastolic index were monitored continuously during supine rest and head up tilt before and one hour after an oral dose of 60 mg pyridostigmine. RESULTS: There was only a modest non-significant increase in supine blood pressure and PRI. In contrast, acetylcholinesterase inhibition significantly increased orthostatic blood pressure and PRI and reduced the fall in blood pressure during head up tilt. Orthostatic heart rate was reduced after the treatment. The improvement in orthostatic blood pressure was associated with a significant improvement in orthostatic symptoms. CONCLUSIONS: Acetylcholinesterase inhibition appears effective in the treatment of neurogenic orthostatic hypotension. Orthostatic symptoms and orthostatic blood pressure are improved, with only modest effects in the supine position. This novel approach may form an alternative or supplemental tool in the treatment of orthostatic hypotension, specially for patients with a high supine blood pressure.

Controlled intermittent asystole cardiac therapy induced by pharmacologically potentiated vagus nerve stimulation in normal and hibernating myocardium.

BACKGROUND: Pharmacologically potentiated electrical stimulation of the right vagus nerve achieves controlled intermittent asystole cardiac therapy. The present study examined pathophysiologic consequences of repetitive intermittent asystoles on contractile function, myocardial blood flow, and vagus nerve function and morphology. METHODS: Open-chest anesthetized canines, with either normal left anterior descending (LAD) coronary arteries (n = 8) or severely stenotic LADs (n = 8), received pharmacologic pretreatment with pyridostigmine (0.5 mg/kg), propranolol (80 microg/kg), and verapamil (50 microg/kg) before vagus nerve stimulation. Time-matched control animals with normal (n = 4) or severely stenotic LADs (n = 6) received drugs but no vagus nerve stimulation. The vagus nerve was stimulated for 12 seconds ("on") and rested for 15 seconds ("off"). This algorithm was repeated for 15 on-off cycles, simulating using controlled intermittent asystole during the placement of 15 sutures in a distal coronary anastomosis. This 15-cycle sequence was repeated twice more, simulating a three-vessel bypass. RESULTS: Normal coronary arteries: Ninety minutes after three sets of controlled intermittent asystole, LAD blood flow was unchanged from base line (36.6 +/- 4.5 versus 33.0 +/- 4.2 mL/min, p = 0.4), and global left ventricular performance (impedance catheter, end-systolic pressure-volume relations) was similar to baseline (7.4 +/- 1.2 versus 7.2 +/- 1.0 mm Hg/mL, p = 0.1). Left anterior descending coronary artery stenosis model: Ninety minutes after CIA, there were no significant differences versus control animals in regional LAD blood flow (27 +/- 4 versus 29 +/- 5 mL/min, p = 0.4) or fractional shortening of LAD myocardium (sonomicrometry; 6.2% +/- 1.8% versus 5.4% +/- 1.2%, p = 0.1). Vagus nerve conduction and morphology were unchanged from baseline. CONCLUSIONS: Repetitive controlled intermittent asystole does not impair poststimulation coronary blood flow, cardiac contractile function, or vagus nerve function. Controlled intermittent asystole may be useful to facilitate off-pump or endoscopic coronary artery bypass grafting.

Complete inhibition of hypothalamic somatostatin activity is only partially responsible for the growth hormone response to strenuous exercise.

The aim of this study was to investigate whether growth hormone (GH) release during strenuous exercise (EX) is due to complete inhibition of hypothalamic somatostatin (SS) activity. Eight healthy male subjects (age, 22.1 +/- 2.2 years; body mass index [BMI], 22.2 +/- 2.5 kg/m(2); maximum oxygen consumption [Vo(2)max], 52.2 +/- 1.5 mL/min/kg [mean +/- SD]) were exposed to strenuous EX on a cycle ergometer, with and without administration of pyridostigmine (PD), and to administration of PD alone. PD is an acetylcholine-esterase inhibitor that stimulates GH secretion by suppressing hypothalamic SS secretion and unmasking endogenous GH-releasing hormone (GHRH) tone. Serial blood samples in the fasted state were taken immediately before the start of each trial, and at appropriate intervals over 2 hours. GH responses were calculated as area under the response curve (AUC) by trapezoidal integration. The mean peak serum GH level to PD alone was 18.3 microg/L (range, 0.3 to 40.9), which was significantly lower than to EX alone: 64.1 microg/L (range, 30.5 to 90.5), and to the combined administration of PD and EX (PD+EX): 79.8 microg/L (range, 37.7 to 98.2) (P <.05). The arithmetic sum of the individual peak levels of 82.4 microg/L was not different from the mean peak level to PD+EX: 79.8 microg/L. AUC (mean +/- SEM) to PD alone (1,721 +/- 358 microg/L x 180 min) was not significantly different from that to EX alone (2,472 +/- 408 microg/L x 120 min), but was significantly lower than that to PD+EX: 3,526 +/- 752 (P <.05). Although the latter AUC was 6% smaller than the AUC obtained by arithmetic addition (3,747 +/- 706), this difference was not statistically significant. In conclusion, the additive effect between PD and EX indicates that PD and EX act independently in evoking GH responses to strenuous EX. Therefore, GH responses to strenuous EX are only partially due to complete inhibition of hypothalamic SS. Additional potentiating factors, such as activation of endogenous GHRH and ghrelin must be operative.

Altered hypothalamic cholinergic responses in patients with nonulcer dyspepsia: a study of pyridostigmine-stimulated growth hormone release.

OBJECTIVE: Acetylcholine plays a central and peripheral role in regulating gastric motility. In the hypothalamus, it is a key neuroendocrine modulator; acting through somatostatin, it brings about the release of growth hormone (GH). We measured hypothalamic cholinergic receptor sensitivity in patients with nonulcer dyspepsia (NUD) by examining GH release in response to cholinergic challenge. METHODS: Forty patients with NUD and 40 healthy comparison subjects were administered pyridostigmine (the acetylcholinesterase inhibitor, 120 mg), and GH release over a 3-h period was monitored. RESULTS: Calculating response as the maximum GH relative to baseline (delta GH), the mean +/- SEM response in the patients was 11.9 +/- 1.9 U/L and in the healthy subjects 6.7 +/- 0.7 mU/L (t = 2.1, df = 78, p = 0.03). Helicobacter pylori status had no appreciable impact on GH response with H. pylori-positive patients having a mean response of 10.5 +/- 2.1 mU/L and negative patients a mean response of 13.2 +/- 3.4 mU/L. Overall, patients with NUD release more GH in response to pyridostigmine challenge than healthy subjects. CONCLUSIONS: Patients with NUD may have a pathophysiological disturbance involving central cholinergic systems.

A dose-response study of anticholinesterase drugs on contractile and phosphatidylinositol responses of rat trachea.

UNLABELLED: We investigated whether anticholinesterase drugs in large doses inhibit muscarinic receptors of airway smooth muscle. In vitro measurements of isometric tension and [(3)H]inositol monophosphate (IP(1)) that formed were conducted by using rat tracheal rings or slices. Neostigmine and pyridostigmine caused muscular contraction and IP(1) accumulation in small doses (10 microM and < or = 100 microM, respectively), but they attenuated muscular contraction and IP(1) accumulation in larger doses (1000 microM). Edrophonium did not affect the smooth muscle tone and IP(1) levels. Neostigmine, pyridostigmine, and edrophonium attenuated the carbachol (5.5 microM)-induced smooth muscle contraction and IP(1) accumulation, when administered in large doses (1000 microM). The attenuation of contraction by neostigmine at large doses was not affected by methoctramine, an M(2) muscarinic receptor antagonist, but was reversed by washing with fresh Krebs-Henseleit solution. The results suggest that anticholinesterase drugs have dual effects on the tension and phosphatidylinositol responses of rat trachea. Large doses of anticholinesterase drugs cause airway smooth muscle relaxation, which may be seen in patients with myasthenia gravis who have received excessive anticholinesterase therapy. IMPLICATIONS: Neostigmine and pyridostigmine, but not edrophonium, have dual effects on the tension and phosphatidylinositol responses of rat trachea. Large doses of anticholinesterase drugs cause airway smooth muscle relaxation, which may be seen in patients with myasthenia gravis who have received excessive anticholinesterase therapy.

Effects of inescapable stress and treatment with pyridostigmine bromide on plasma butyrylcholinesterase and the acoustic startle response in rats.

Pyridostigmine bromide (PB) is a reversible, peripherally active inhibitor of acetylcholinesterase (AChE) activity, and is recommended by the military as a pretreatment against potential nerve gas exposure. Recent evidence suggests that exposure to inescapable stressors allows PB to cross the blood-brain barrier, and thereby affect central AChE activity in mice. Here, we evaluated the functional impact of a stress/PB treatment interaction on acoustic startle responding and plasma butyrylcholinesterase (BuChE) activity in male Sprague-Dawley rats. To model the treatment protocol used by the military, PB was delivered in the drinking water of rats for 7 consecutive days. The morning after the start of PB treatment, and for the next 6 days, half the rats were exposed to 1 h of supine restraint stress. We therefore employed a 2 x 2 (stress x PB treatment) between-groups design. Exposure to supine stress alone induced a persistent decrease in plasma BuChE activity. Further decreases in BuChE activity were not observed in rats exposed to supine restraint and PB treatment. Exposure to stress also induced an exaggerated startle response, evident on the last day of stress and 24 h after stressor cessation. Treatment with PB alone produced an exaggerated startle response over the same time period, albeit to a lesser degree. Although treatment with PB concurrent with stress did not produce further changes in either BuChE activity or acoustic startle responding, stress-induced alterations in drinking behavior (and thereby the dose of PB ingested) may have affected these results. Persistent stress-induced reductions in BuChE activity may increase the risk of adverse reactions to cholinomimetics.

Dopaminergic and cholinergic involvement in the inhibitory effect of dexamethasone on the TSH response to TRH.

BACKGROUND: Glucocorticoid administration is associated with reduced basal thyroid-stimulating hormone (TSH) levels and a blunted TSH response to thyrotropin-releasing hormone (TRH), despite thyroid hormone levels within the normal range. In light of the inhibitory effect of somatostatin and dopamine on TSH secretion, we examined whether this condition is caused by glucocorticoids through an increased hypothalamic somatostatinergic and/or dopaminergic inhibitory control of TSH. We measured the TSH response to TRH and serum-free T4 and T3 levels. The study group comprised 18 normal men (age 24-35) within 10% of the ideal body weight, randomly divided into 3 groups of six. METHODS: We used the antidopaminergic agent metoclopramide (MCP) and the acetylcholinesterase inhibitor pyridostigmine, which enhances acetylcholine and thus inhibits hypothalamic somatostatin release. Subjects from group 1 were tested with TRH (20 micrograms in an intravenous bolus) after placebo, dexamethasone (dex) (2 mg/day in 4 divided doses for 3 days before the experimental day), or dex plus pyridostigmine (120 mg p.o.). Subjects from group 2 were tested with TRH after placebo, dex, or dex plus MCP (2.5 mg in an i.v. bolus injection). Subjects from group 3 were tested with TRH after placebo, dex, or dex plus pyridostigmine plus MCP. RESULTS: In all subjects from groups 1, 2, and 3, TRH-induced TSH rise was significantly lower after dex than after placebo treatment. Neither pyridostigmine nor MCP, given alone, changed the TSH response to TRH after dex treatment. In contrast, the concomitant administration of MCP and pyridostigmine significantly enhanced the TRH-induced TSH rise in dex-treated subjects and made the TSH response to TRH similar to that observed in the TRH plus placebo test. CONCLUSIONS: These data indicate that enhanced-hypothalamic somatostatinergic and dopaminergic inhibitory activities are involved in the mechanism underlying the reduced TSH response to TRH induced by glucocorticoid treatment.

Mechanisms underlying the negative growth hormone (GH) autofeedback on the GH-releasing effect of hexarelin in man.

The growth hormone (GH) response to GH-releasing hormone (GHRH) is strongly inhibited by previous administration of recombinant human GH (rhGH), likely as a consequence of a somatostatin-mediated GH negative autofeedback. Hexarelin (HEX), a synthetic hexapeptide belonging to the GH-releasing peptide (GHRP) family, possesses a GH-releasing activity greater than that of GHRH both in animals and in man. The mechanism of action of GHRPs is yet to be completely clarified, although concomitant actions at the pituitary and hypothalamic level have been hypothesized. To further clarify the mechanisms of action underlying the GH-releasing activity of HEX, in six normal young volunteers we studied the effects of rhGH (2 U intravenously [IV]) on the GH response either to GHRH (2 microg/kg IV) or to HEX (2 microg/kg IV) alone or combined with GHRH and/or pyridostigmine ([PD], 120 mg orally). The GH-releasing effect of HEX was higher than that of GHRH (area under the curve [AUC], 2,200.8 +/- 256.9 v 792.2 +/- 117.6 microg/L/h, P < .001), whereas combined administration of the two substances induced a true synergistic effect, with GH release after HEX plus GHRH (4,259.2 +/- 308.0 microg/L/h) being higher (P < .02) than the arithmetic sum of the GH increases induced by each compound separately administered. After rhGH administration, the GH-releasing effect of HEX was blunted (1,468.9 +/- 193.7 microg/L/h, P < .04; inhibition of 32.1%), whereas that of GHRH was nearly abolished (102.0 +/- 7.8 microg/L/h, P < .02; inhibition of 86.1%). The GH response to combined administration of HEX and GHRH was also blunted by the previous rhGH bolus (3,070.6 +/- 481.8 microg/L/h, P < .02; inhibition of 26.7%). PD did not modify the GH-releasing effect of HEX either alone (2,456.8 +/- 317.5 microg/L/h) or combined with GHRH (4,009.1 +/- 360.8 microg/L/h). rhGH was again able to blunt the GH response to HEX combined with PD (1,619.3 +/- 237.9 microg/L/h, P < .02), but failed to modify the GH response to HEX combined with GHRH and PD (4,548.4 +/- 698.0 microg/L/h). In conclusion, these results demonstrate that rhGH administration only blunts the GH-releasing activity of HEX, but abolishes that of GHRH. The blunting effect of rhGH on the GH response to HEX is probably mediated by a concomitant reduction in the activity of GHRH-secreting neurons and an increase of somatostatinergic tone.

Suppression of TRH-stimulated TSH secretion by glucose-induced hypothalamic somatostatin release.

To determine whether the combined glucose-thyrotropin-releasing hormone (TRH) test can be a useful method for the evaluation of the hypothalamic somatostatinergic activity, we investigated whether TRH-induced thyroid stimulating hormone (TSH) secretion can be suppressed by the oral glucose administration that stimulates the hypothalamic somatostatin (SRIH) secretion. Six tests were performed in ten healthy young men. Test 1: 1 ml of normal saline was intravenously administered at 0 min. Test 2: TRH was administered intravenously at 0 min. Test 3: Glucose, 75 g, was administered orally at -60 min. Test 4: Glucose and TRH were administered as above. Test 5: Pyridostigmine (PST), 120 mg, was given orally at -90 min followed by the administration of GH and TRH as above. Basal TSH levels were suppressed slightly, but significantly. In Test 3 compared to those observed in Test 1. The oral glucose administration also significantly suppressed TRH-stimulated TSH response by 27-35% between 40 min and 80 min in Test 4. In contrast, the pretreatment with PST completely reverted the suppressive effect of glucose on TRH-stimulated TSH response in Test 5. These data suggest that the increased hypothalamic SRIH secretion induced by oral glucose administration can suppress TRH-stimulated TSH response in normal men, and the combined glucose-TRH test can be a useful method to evaluate the hypothalamic somatostatinergic activity.

Adrenergic and cholinergic involvement in basal and growth hormone-releasing hormone-stimulated growth hormone secretion in glucocorticoid-treated rats.

Glucocorticoids are known to inhibit GH secretion via somatostatin. The aim of our study was to elucidate the involvement of somatostatin in the GH-releasing action of the alpha 2 agonist clonidine and the cholinergic agent pyridostigmine in conscious, freely-moving rats chronically treated with dexamethasone. After seven days of chronic glucocorticoid treatment, animals received an i.v. injection of either saline (1 ml/kg) or clonidine (150 micrograms/kg) or pyridostigmine (100 micrograms/kg) at -15 min. Three blood samples were then drawn (-10 min, -5 min, and 0 min) to assess the GH response to either clonidine or pyridostigmine alone. After the 0 min sample, saline (1 ml/kg) or GNRH (500 ng/kg) was injected i.v. and additional blood samples were drawn from 5 to 30 min. The GH response to clonidine alone or combined with GNRH in rats treated with dexamethasone was significantly lower (p < 0.05) as compared to vehicle-treated rats. The GH response to pyridostigmine alone or combined with GNRH did not significantly differ between vehicle- and dexamethasone-treated rats. These data suggest that in the rat the mechanism of action of clonidine is mainly to stimulate endogenous GNRH secretion, while pyridostigmine appears to predominantly act by decreasing hypothalamic somatostatin.

Regulation of hypothalamic somatostatin by glucocorticoids.

Glucocorticoids (GCs) play a key role in the physiology of the hypothalamic-somatotroph axis, since these steroids enhance growth hormone (GH) gene transcription and increase GHRH receptor synthesis. However, GC excess inhibits normal growth in all species studied. This is mainly due to the impaired GH secretion observed during hypercortisolism, a situation in which GH responses to a number of stimuli, including GHRH, are blunted. The inhibitory effect of GCs on GH secretion seems to be dependent on enhanced hypothalamic SS secretion. Since SS release is stimulated by beta-adrenergic agonism we tested the possibility that GC inhibition of GH secretion would depend on increased beta-adrenoceptor activity in SS-producing neurons. The experimental design consisted in evaluating the GH response to GHRH in normal subjects after having induced hypercortisolism, with DEX, and blocking beta-adrenoceptors with propranolol (PRO). Moreover, to investigate the specificity of this mechanism, GHRH-induced GH release was tested after inducing hypercortisolism and enhancing alpha 2-adrenergic or muscarinic cholinergic tone, by giving clonidine (CLO) or pyridostigmine (PD), respectively. As expected, nocturnal DEX administration inhibited the GH response to GHRH. In this situation of hypercortisolism, both PRO and CLO, but not PD, were able to reverse the inhibitory effect of DEX on GHRH-elicited release. However, the potentiating effect of these drugs on the GHRH-induced GH secretion was only observed for PRO. These data confirm that GC excess inhibits GH release by increasing hypothalamic SS secretion, and that the mechanism is mediated by GC-induced enhanced beta-adrenergic responsiveness. Therefore, the defective GHRH secretion observed in chronic hypercortisolism must be a consequence of the continuous blockade that SS excess exerts on GHRH-producing neurons. Our postulate agrees with other data in the literature showing that GCs modulate the secretion of some hypothalamic peptides by changing the responsiveness of the producing neurons from alpha 2-adrenoceptors to that of beta-adrenoceptors.

Acute toxicity of cyclohexylmethylphosphonofluoridate (CMPF) in rhesus monkeys: serum biochemical and hematologic changes.

Changes in serum biochemical and hematological parameters were studied in 20 male rhesus monkeys following acute poisoning by the organophosphate nerve agent cyclohexylmethylphosphonofluoridate (CMPF or GF). Animals were challenged with 5 x LD50 GF (233 micrograms/kg, IM) following pretreatment with pyridostigmine (0.3-0.7 mg/kg per 24 h) and treated with atropine (0.4 mg/kg, IM) and either 2-PAM (25.7 mg/kg, IM) or H16 (37.8 mg/kg, IM) at the onset of clinical signs or at 1 min after exposure. Muscle fasciculations, tremors, or convulsions occurred in 19 of 20 animals. Serum biochemical and hematologic parameters were analyzed 2 days and 7 days after exposure and compared to pre-exposure baseline values. Significant increases in creatine kinase (CK), lactate dehydrogenase (LD), aspartate transaminase (AST), alanine transaminase (ALT) and potassium ion (K+), associated with damage to striated muscle and metabolic acidosis, occurred in both oxime-treated groups 2 days after exposure. Total protein, albumin, red blood cell (RBC) count, hemoglobin concentration (Hb) and hematocrit (Hct), were decreased in both oxime-treated groups at 7 days. The results demonstrate that animals exposed to a single high dose of GF and treated with standard therapy exhibit changes in serum biochemical and hematological indices directly and indirectly associated with their clinical presentations.