Nitric oxide reduces the thermogenic changes induced by lateral hypothalamic lesion

The experiment described here tests the effect of intracerebroventricular (icv) injection of nitric oxide (NO) precursors, such as L-arginine (L-arg) and nitroprusside (NP), on the thermogenic changes induced by lesion of the lateral hypothalamus (LH). The firing rate of the nerves innervating interscapular brown adipose tissue (IBAT), along with IBAT and colonic temperatures (T_IBAT and T_C) were monitored in urethane-anesthetized male Sprague-Dowley rats lesioned in the LH. These variables were measured before and after and icv injection of 4 μmol L-arg or 400 nmol NP. The same variables were also monitored in: a) lesioned rats with icv administration of saline; b) sham-lesioned animals with icv injection of L-arg or NP; c) sham-lesioned rats with icv injection of saline. The results show that L-arg or NP injection reduces the increases in firing rate, T_IBAT, and T_C induced by LH lesion. These findings suggest that NO plays a key role in the thermogenic changes following LH lesion.     

Prostaglandin E1 or E2 (PGE1, PGE2) prevents premature luteolysis induced by progesterone given early in the estrous cycle in ewes

The objective of this study was to determine whether prostaglandin E₁ (PGE₁) or prostaglandin E₂ (PGE₂) prevents premature luteolysis in ewes when progesterone is given during the first 6 days of the estrous cycle. Progesterone (3 mg in oil, im) given twice daily from Days 1 to 6 (estrus = Day 0) in ewes decreased (P < 0.05) luteal weights on Day 10 postestrus. Plasma progesterone concentrations differed (P < 0.05) among the treatment groups; toward the end of the experimental period, concentrations in jugular venous blood decreased (P < 0.05) compared with the other treatment groups. Plasma progesterone concentrations in ewes receiving PGE₁ or PGE₁ + progesterone were greater (P < 0.05) than in vehicle controls or in ewes receiving PGE₂ or PGE₂ or PGE₂ + progesterone. Chronic intrauterine treatment with PGE₁ or PGE₂ prevented (P < 0.05) decreases in plasma progesterone concentrations, luteal weights, and the proportion of luteal unoccupied and occupied LH receptors on Day 10 postestrus in ewes given exogenous progesterone, but did not affect (P > 0.05) concentrations of PGF_2α in inferior vena cava blood. Progesterone given on Days 1 to 6 in ewes advanced (P < 0.05) increases in PGF_2α in inferior vena cava blood. We concluded that PGE₁ or PGE₂ prevented progesterone-induced premature luteolysis by suppressing loss of luteal LH receptors (both unoccupied and occupied).     

PGE1 metabolism by the perfused rat liver

The hepatic and biliary metabolites of PGE₁ have been isolated and identified after infusions of PGE₁ into isolated rat liver preparations. The results demonstrate that in general PGE₁ undergoes metabolism similar to that of PGE₂ in the rat and reveals the possibility of a selective PG metabolite transport system across the biliary canalicular membrane.     

Utility of d4-PGE2 as an internal standard to quantify endogenous levels of PGE1, PGE2, 190H PGE1 and 190H PGE2 in human seminal fluid by GC-MS-SIM

Four prostaglandins-PGE₁, PGE₂, 190H PGE₁ and 190H PGE₂-were quantified in human seminal fluid by GC-MS-SIM using only the internal standard, d₄-PGE₂. Methods and calculations were developed to minize errors inherent in using only one internal standard for quantifying four closely related prostaglandins. Preliminary data concerning the statistical significance of the differences found between PGE and 190H PGE levels in fertile, azospermic and oligospermic men are reported.     

A comparison of uterine contraction induced by PGE1 and oxytoxin in Ca-gree solution

The contraction of the rat uterus incubated in Ca-free EDTA-containing solution in response to PGE₁, oxytoxin and vanadate has veen ivestigated in order to examine the mechanism of the release of Ca from intracellular stores. The results obtained show that PGE₁ evoked a sustained contraction the magnitude of which diminishes slightly after successive additions of PGE₁ but not after long exposure to Ca-free medium. Oxytoxin induced two different contractions: one of them was transient and observed only after incubating for 5 min in Ca-free solution; the other remained constant during prolonged incubation in Ca-free medium. Vanadate, an inhibitor of Ca-ATPase, induced sustained contraction after prolonged exposure to Ca-free medium, and isoprenaline, which stimulates Ca re-uptake by intracellular organelles, counteracted the sustained contractile response induced by the three agonists.     

A dose-response comparison of PGA2, PGE1 and PGE2 using the phenylephrine-stimulated perfused oviduct of the rabbit

The phenylephrine-stimulated perfused oviduct of the rabbit was evaluated as a model for studying the activity of prostaglandins that produce inhibition of the oviducal smooth muscle. Elevation of the normal “tone” of the oviduct by perfusing phenylephrine through the lumen permitted quantitation of the responses to PGA₂, PGE₁ and PGE₂ by measuring the magnitude of the inhibitory response produced by the agents. PGE₂ was relatively more potent, efficacious and specific for the oviduct than PGA₂ or PGE₁. It was concluded that the model was suitable for comparative dose-response studies of PGA₂, PGE₁ and PGE₂ and their analogs.     

Selective antagonism of the systemic vasodepressor response to PGE1 by d,1–11, 15-bisdeoxy PGE1

Several bisdeoxy PGE₁ analogs are potent, competitive antagonists of PGE₁-induced colonic contractions in the gerbil. The efficacy of these analogs in antagonizing PGE₁-mediated systemic vasodepression has not been previously demonstrated. In this study, serial doses of PGs were administered before, during and after infusion of d,1–11, 15-bisdeoxy PGE₁. Bolus injections of PGE₁ (3.0 μk/kg), PGE₂ (3.0 μg/kg) and PGI₂ (0.3 μg/kg) were administered via the right external jugular vein to male Wistar rats. PGE₁, PGE₂ and PGI₂ decreased systemic arterial pressure 41%, 38% and 38%, respectively. The PGE₁ analog was infused (200 μg/kg/min) through the right common carotid artery. The analog itself had no effect on mean systemic arterial pressure, but maximum reversible inhibition (51%) of PGE₁-mediated vasodepression occurred following a 50 minute infusion. No significant effect of the PGE₁ analog was observed on PGE₂ or PGI₂-mediated vasodepression. These data demonstrate the ability to antagonize PGE₁-mediated vasodepression, and to differentiate the vascular responses to PGE₁ and PGE₂ or PGI₂.     

Influence of polyphloretin phosphate (PPP) on rat-paw oedema induced by prostaglandin E1 (PGE1)

As a result of pretreatment with the known prostaglandin antagonist polyphloretin phosphate (50, 100 or 200 mg/kg i.v.), there is a statistically significant decrease in the magnitude of the oedema induced in Sprague-Dawley CFY rats by the subplantar administration of 1 μg prostaglandin E₁.     

Inotropic effect of PGE1 and PGE2 on isolated rat atria. Influence of adrenergic mechanisms

The effects of a wide range of PGE₁ and PGE₂ concentrations on the isometric developed tension of isolated rat atria beating spontaneously or paced at a fixed rate, were explored. PGE₁ only produced a negative inotropic effect (NIE), whereas PGE₂ elicited a biphasic inotropic action; negative at low concentrations and positive (PIE) at higher ones. Phenoxybenzamine and phentolamine failed to modify either the NIE or the PIE, but subthreshold exogenous norepinephrine abolished the NIE, suggesting a presynaptic inhibitory effect of PGEs on the adrenergic neurotransmitter release. Auricles pretreated with subthreshold norepinephrine react with a PIE to PGE₁, but not to PGE₂. On the contrary in the presence of subthreshold methoxamine the PIE of PGE₂ was increased whereas the action of PGE₁ was not modified.     

The effects of prostaglandins PGE1, PGE2, PGF1a, and PGF2a on canine synovial perfusion

In these experiments we have examined the effects of PGE₁, PGE₂, PGF_1α and PGF_2α on synovial perfusion in the normal canine synovial microcirculation. The effects of the drugs on synovial perfusion were determined indirectly from the changes produced in the rate of clearance of ¹³³Xenon from the joint by their intra-articular injection. Prostaglandins PGE₁ and PGE₂ were found to be strongly vasodilator with PGE₁ being the more active. PGF_1α appeared to have little or no vasoactive properties in doses up to 1 ugm. (2.8 × 10^−5M) in our I preparation while PGF_2α was vasodilator at this high dosage only. Neither SC19920 nor diphloretin phosphate antagonised the effects of PGE₁ in these experiments.     

Calcium requirement of uterine contraction induced by PGE1: Importance of intracellular calcium stores

The contraction of the rat uterus in response to PGE₁ in high K⁺ medium and in Ca-free solution which contained EDTA has been investigated in order to examine whether excitation-contraction coupling involves the release of Ca from an intracellular store.In uterus maximally contracted by K⁺, cumulative concentrations of PGE₁ (1.25 – 20 ng/ml) caused maintained concentration-dependent contraction. PGE₁ induced sustained contraction of rat uterus in Ca-free medium after incubation with 3mM EDTA for 50 min. In these conditions the involvement of extracellular Ca is highly unlikely. The PGE₁-induced contraction could be repeated without exposure to external Ca ions and with only slight reduction in magnitude. The PGE₁ concentrations required to elicit uterine contraction in Ca-free solution were about 1000 times higher than the effective doses in KCl-depolarized uterus.In conclusion, the present investigation shows that Ca influx is not essential for PGE₁-induced contraction of rat uterus, although extracellular Ca enhances it presumably by increasing the free Ca levels in the cytosol.     

Inhibition of an IUD-induced precocious luteolysis by prostaglandin E1 (PGE1) in sheep

Fifteen ewes were assigned as they came into estrus to one of three randomized treatment groups: 1. Sham IUD + Vehicle, 2. IUD + Vehicle or 3. IUD + PGE₁ in vehicle. An IUD was inserted adjacent to the luteal-bearing ovary on day 3 postestrus. Prostaglandin E₁ (500 μg) in vehicle (Na₂CO₃) or vehicle was given intrauterine through an indwelling uterine cannula every four hours from day 3 postestrus until ewes returned to estrus. Precocious estrus was induced in both the sham IUD groups receiving vehicle. Prostaglandin E₁ prevented an IUD-induced premature luteolysis based on daily concentrations of progesterone in peripheral blood and the interestrous interval. It is concluded that an IUD-induced premature luteolysis is not necessarily via physical distention by the IUD. It is also concluded that chronic intrauterine infusions of PGE₁ can prevent an IUD-induced premature luteolysis.     

The effect of PGE2 on the activation of quiescent lung fibroblasts

The effect of prostaglandin E₂ (PGE₂) on fibroblast proliferation was examined. The presence of PGE₂ for 24 h inhibited the growth of quiescent cells stimulated with serum, platelet-derived growth factor and macrophage-derived factors. Maximal inhibition of nuclear labeling with [³H]thymidine occurred at concentrations greater than 10⁻⁷ M. The inhibitory effect of PGE₂ was less potent in exponentially growing cells and was not the result of conversion of PGE₂ to PGA₂ during incubation in growth medium. The G₁ phase was determined to be 12–14 h in untreated cultures. The extent of growth inhibition by PGE₂ was similar with addition of PGE₂ at 0, 3, 6, or 9 h following restimulation of quiescent cell cultures. Approximately 25% of the cells that enter S phase are refractory to PGE₂-induced growth inhibition. Short-term exposure to PGE₂ (5 min and 30 min) caused substantial growth inhibition. The serum-induced proliferation was also inhibited by the cAMP analogue, dibutyrl cAMP. Our results suggest that PGE₂ affects a distinct subpopulation of cells. Restimulation of quiescent cells treated with PGE₂ for 24 h, indicated that release from PGE₂ exposure is associated with prolongation of the G₁ phase of the cell cycle.     

Effects of intracerebroventricular administration of PGE1, E2 and F2α on electrically induced convulsions in mice

Intracerebroventricular administration of prostaglandins E₁ or E₂ was shown to block, while PGF_2α increased the incidence of tonic convulsion due to electroshock in mice. The Prostaglandins were administered intracerebroventricularly (i.c.v.) to conscious mice by a modification of Haley and McCormick's method (1) prior to a transcorneal maximal electroshock (MES) or a transcorneal supra-maximal electroshock (SMES). PGE₁ and PGE₂ i.c.v. blocked the tonic hindlimb extension (THE) and protected the animals from death induced by MES with ED₅₀'s for PGE₁ and PGE₂ for inhibition of the THE of 6.6 (4.3–12.0) μg/mouse i.c.v. and 13.3 (8.9–22.4) μg/mouse i.c.v. respectively. When PGE₂ was administered intraperitoneally (i.p.) in doses as high as 4.0 mg/kg it did not block the THE. However, the duration of the THE as well as the mortality were reduced by doses of 0.5–4.0 mg/kg PGE₂ i.p.. Both PGE₁ and PGE₂ were shown to cause a dose related significant (p<.001) decrease in the duration of the THE with SMES in doses of 1–10 μg/mouse i.c.v. for PGE₁ and 2–40 μg/mouse i.c.v. for PGE₂. PGF_2α, administered i.c.v. prior to a transcorneal electroshock equivalent to a current at the ED₁ level, increased the incidence of the THE as well as the mortality in doses of 20–50 μg/mouse.     

Interactions of prostaglandin E1 (PGE1) and LRH on anterior pituitary function

Numerous biochemical pathways influence the synthesis and release of anterior pituitary hormones. Releasing factors extracted from the hypothalamus and prostaglandins (PGs) appear to alter a common biochemical activity, adenyl cyclase, in pituitary cells. Luteinizing hormone releasing hormone (LRH), prostaglandin (PGE₁), 7 oxa-13-prostynoic acid and cycloheximide were tested for individual and interacting effects on the in vitro release of FSH, LH and prolactin from hemipituitaries of 15 day old female rats. LRH (10 ng/ml) consistently released both LH and FSH in all in vitro experiments and inhibited prolactin release in 1 of 2 experiments. Lower concentrations (5 and 1 ng/ml) also stimulated LH and FSH release but did not influence prolactin release. Concurrent depletion of stored LH and FSH in the gland was observed. PGE₁ in a 6.5 hour incubation increased the storage of LH within the gland in the absence of LRH. In a 1.5 hour incubation in the presence of LRH, storage of LH was also increased. PGE₁ had no effect on LH and FSH release; however, in 1 of 2 experiments it stimulated prolactin release in the absence of LRH. Prostynoic acid stimulated LH and FSH release but did not synergize with LRH action in the same tissue. Cycloheximide did not affect LH release during the first 30 minutes of incubation; however, the release during the subsequent 1 hour was significantly inhibited. Similar tissue also exposed to cycloheximide was still responsive to LRH during the latter 1 hour incubation period. Cycloheximide had no effect on prolactin storage and release from the same tissue.     

Interleukin-1 and tumor necrosis factor are not synergistic for human synovial fibroblast PLA2 activation and PGE2 production

Patterns of arachidonic acid release and metabolism were altered in human synovial fibroblasts following exposure to cytokines. Recombinant interleukin-1 induced an approximate 3-fold in crease in [³H]-AA release, a 7-fold increase in PGE₂ production and a 2-fold increase in PLA₂ activity in human synovial fibroblasts. Recombinant tumor necrosis factor induced similar responses, however, the magnitude was less than that mediated by interleukin-1. A combination of the two cytokines had an additive effect on [³H]-AA release and PLA₂ activity while PGE₂ production was similar to that detected using interleukin-1 alone. [³H]-AA, was released in substantial amounts when sodium fluoride was used as a stimulus but PGE₂ was not. These data show that tumor necrosis factor and interleukin-1 can both activate synovial cell PLA₂ and induce generation of PGE₂, but act in an additive rather than a synergistic fashion. Furthermore, the data show that PGE₂ production is not always concordant with [³H]-AA release, suggesting that appropriate enzyme(s) must be activated.     

Interactions of prostaglandin E1 (PGE1) and LRH on anterior pituitary function

Numerous biochemical pathways influence the synthesis and release of anterior pituitary hormones. Releasing factors extracted from the hypothalamus and prostaglandins (PGs) appear to alter a common biochemical activity, adenyl cyclase, in pituitary cells. Luteinizing hormone releasing hormone (LRH), prostaglandin (PGE₁), 7 oxa-13-prostynoic acid and cycloheximide were tested for individual and interacting effects on the $\text{in vitro}$ release of FSH, LH and prolactin from hemipituitaries of 15 day old female rats. LRH (10 ng/ml) consistently released both LH and FSH in all $\text{in vitro}$ experiments and inhibited prolactin release in 1 of 2 experiments. Lower concentrations (5 and 1 ng/ml) also stimulated LH and FSH release but did not influence prolactin release. Concurrent depletion of stored LH and FSH in the gland was observed. PGE₁ in a 6.5 hour incubation increased the storage of LH within the gland in the absence of LRH. In a 1.5 hour incubation in the presence of LRH, storage of LH was also increased. PGE₁ had no effect on LH and FSH release; however, in 1 of 2 experiments it stimulated prolactin release in the absence of LRH. Prostynoic acid stimulated LH and FSH release but did not synergize with LRH action in the same tissue. Cycloheximide did not affect LH release during the first 30 minutes of incubation; however, the release during the subsequent 1 hour was significantly inhibited. Similar tissue also exposed to cycloheximide was still responsive to LRH during the latter 1 hour incubation period. Cycloheximide had no effect on prolactin storage and release from the same tissue.     

The effects of PGF1α, PGE2 and 16,16 dimethyl PGE2 on gastric emptying and small intestinal transit in rat

Prostaglandins are well known for their ability to stimulate contraction in gastrointestinal smooth muscle, yet very little information is available on how their activity affects propulsion . Thus, studies were undertaken to determine the effect of various prostaglandins on qastric emptying (GE) and small intestinal transit (SIT) in unanesthetized fasted rats. Rats were treated with intravenous, subcutaneous, or oral PGF_2α, PGE₂, or 16,16 dimethyl PGE₂ at various doses, followed 1 (intravenous), 20 (subcutaneous) or 10 (oral) mins later by intragastric ⁵¹Cr oxide in black ink. Forty-five mins later, rats were sacrificed by CO₂ asphyxiation, the pylorus clamped, and the gut excised. SIT was expressed as the percent of intestinal length traveled by the most distal portion of ink. GE was expressed as the percent of the ⁵¹Cr emptied into the intestines. If GE was affected by prostaglandin treatment, the experiments were repeated with rats pre-implanted with duodenal cannula. This preparation allowed the visual transit marker to be deposited directly into the dueodenum, thus avoiding acceleration or delay of SIT caused by fluctuations in GE. The results of these studies show that: (1) intravenous 16,16 dimethyl PGE₂ (5–50 μg/kg), but not PGF_2α or PGE₂, accelerates GE and delays SIT; (2) oral prostaglandin administration increases SIT; (3) oral 16,16 dimethyl PGE₂ delays GE; (4) subcutaneous 16,16 dimethyl PGE₂ accelerates, has no effect upon, or delays GE depending upon dose, but accelerates SIT at all doses tested; and (5) subcutaneous PGE₂ accelerates SIT while PGF_2α does not. Thus, the effect of prostaglandins on GE and SIT depends upon the dosage and route of administration as well as type of prostaglandin used.     

P2X2, P2X2–2 and P2X5 receptor subunit expression and function in rat thoracolumbar sympathetic neurons

The present study investigated the pharmacological properties of excitatory P2X receptors and P2X(2) and P2X(5) receptor subunit expression in rat-cultured thoracolumbar sympathetic neurons. In patch-clamp recordings, ATP (3-1000 microM; applied for 1 s) induced inward currents in a concentration-dependent manner. Pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS; 30 microM) counteracted the ATP response. In contrast to ATP, alpha,beta-meATP (30 microM; for 1 s) was virtually ineffective. Prolonged application of ATP (100 microM; 10 s) induced receptor desensitization in a significant proportion of sympathetic neurons in a manner typical for P2X(2-2) splice variant-mediated responses. Using single-cell RT-PCR, P2X(2), P2X(2-2) and P2X(5) mRNA expression was detectable in individual tyrosine hydroxylase-positive neurons; coexpression of both P2X(2) isoforms was not observed. Laser scanning microscopy revealed both P2X(2) and P2X(5) immunoreactivity in virtually every TH-positive neuron. P2X(2) immunoreactivity was largely distributed over the cell body, whereas P2X(5) immunoreactivity was most distinctly located close to the nucleus. In summary, the present study demonstrates the expression of P2X(2), P2X(2-2) and P2X(5) receptor subunits in rat thoracolumbar neurons. The functional data in conjunction with a preferential membranous localization of P2X(2)/P2X(2-2) compared with P2X(5) suggest that the excitatory P2X responses are mediated by P2X(2) and P2X(2-2) receptors. Apparently there exist two types of P2X(2) receptor-bearing sympathetic neurons: one major population expressing the unspliced isoform and another minor population expressing the P2X(2-2) splice variant.