(1/1305) Transcriptional regulation of alpha1-adrenoceptor gene in the rat liver during different phases of sepsis.
Changes in alpha1-adrenoceptor (alpha1AR) gene expression in the rat liver during different phases of sepsis were studied. Sepsis was induced by cecal ligation and puncture (CLP). Septic rats exhibit two metabolically distinct phases: an initial hyperglycemic phase (9 h after CLP, early sepsis) followed by a hypoglycemic phase (18 h after CLP; late sepsis). The [3H]prazosin binding studies show that the density of alpha1AR was increased by 30% during the early phase while it was decreased by 24% during the late phase of sepsis. Western blot analyses reveal that alpha1AR protein level was elevated by 48% during early sepsis but was decreased by 55% during late sepsis. Northern blot analyses depict that the steady-state level of alpha1bAR mRNA was enhanced by 21% during the early phase but was declined by 29% during the late phase of sepsis. Nuclear run-off assays show that the transcription rate of alpha1bAR gene transcript was increased by 76% during early sepsis while it was decreased by 29% during late sepsis. The actinomycin D pulse-chase studies indicate that the half-life of alpha1bAR mRNA remained unaffected during the early and the late phases of sepsis. These findings demonstrate that during the early phase of sepsis, the increase in the rate of transcription of alpha1bAR gene paralleled with the elevations in the alpha1bAR mRNA abundance and alpha1AR protein level, while during the late phase of sepsis, the decrease in the rate of transcription of alpha1bAR gene coincided with the declines in the alpha1bAR mRNA abundance and the alpha1AR protein level in the rat liver. These observations indicate that the altered expression of alpha1AR genes in the rat liver during the progression of sepsis was regulated transcriptionally. (+info)
(2/1305) Expression of alpha2-adrenergic receptors in rat primary afferent neurones after peripheral nerve injury or inflammation.
1. Immunocytochemistry with polyclonal antibodies directed against specific fragments of intracellular loops of alpha2A- and alpha2C-adrenergic receptors (alpha2A-AR, alpha2C-AR) was used to explore the possibility that expression of these receptors in dorsal root ganglion (DRG) neurones of rat alters as a result of peripheral nerve injury or localized inflammation. 2. Small numbers of neurones with positive alpha2A-AR immunoreactivity (alpha2A-AR-IR) were detected in DRG from normal animals or contralateral to nerve lesions. In contrast, after complete or partial sciatic nerve transection the numbers of ipsilateral L4 and L5 DRG somata expressing alpha2A-AR-IR sharply increased (>5-fold). There was no discernible change in the number of DRG neurones exhibiting alpha2A-AR-IR innervating a region in association with localized chemically induced inflammation. 3. After nerve injury, double labelling with Fluoro-Gold, a marker of retrograde transport from transected fibres, or by immunoreactivity for c-jun protein, an indicator of injury and regeneration, suggested that many of the neurones expressing alpha2A-AR-IR were uninjured by the sciatic lesions. 4. In general the largest proportionate increase in numbers of neurones labelled by alpha2A-AR-IR after nerve lesions appeared in the medium-large diameter range (31-40 microm), a group principally composed of cell bodies of low threshold mechanoreceptors. The number of small diameter DRG neurones labelled by alpha2A-AR-IR, a category likely to include somata of nociceptors, also increased but proportionately less. 5. Relatively few DRG neurones exhibited alpha2C-AR-IR; this population did not appear to change after either nerve lesions or inflammation. 6. These observations are considered in relation to effects of nerve injury on excitation of primary afferent neurones by sympathetic activity or adrenergic agents, sympathetically related neuropathy and reports of sprouting of sympathetic fibres in DRG. (+info)
(3/1305) alpha-adrenergic stimulation mediates glucose uptake through phosphatidylinositol 3-kinase in rat heart.
We examined whether insulin and catecholamines share common pathways for their stimulating effects on glucose uptake. We perfused isolated working rat hearts with Krebs-Henseleit buffer containing [2-3H]glucose (5 mmol/L, 0.05 microCi/mL) and sodium oleate (0.4 mmol/L). In the absence or presence of the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin (3 micromol/L), we added insulin (1 mU/mL), epinephrine (1 micromol/L), phenylephrine (100 micromol/L) plus propranolol (10 micromol/L, selective alpha-adrenergic stimulation), or isoproterenol (1 micromol/L) plus phentolamine (10 micromol/L, selective beta-adrenergic stimulation) to the perfusate. Cardiac power was found to be stable in all groups (between 8.07+/-0.68 and 10.7+/-0. 88 mW) and increased (25% to 47%) with addition of epinephrine, but not with selective alpha- and beta-adrenergic stimulation. Insulin and epinephrine, as well as selective alpha- and beta-receptor stimulation, increased glucose uptake (the following values are in micromol/[min. g dry weight]: basal, 1.19+/-0.13; insulin, 3.89+/-0.36; epinephrine, 3.46+/-0.27; alpha-stimulation, 4.08+/-0.40; and beta-stimulation, 3.72+/-0.34). Wortmannin completely inhibited insulin-stimulated and selective alpha-stimulated glucose uptake, but it did not affect the epinephrine-stimulated or selective beta-stimulated glucose uptake. Sequential addition of insulin and epinephrine or insulin and alpha-selective stimulation showed additive effects on glucose uptake in both cases. Wortmannin further blocked the effects of insulin on glycogen synthesis. We conclude that alpha-adrenergic stimulation mediates glucose uptake in rat heart through a PI3-K-dependent pathway. However, the additive effects of alpha-adrenergic stimulation and insulin suggest 2 different isoforms of PI3-K, compartmentation of PI3-K, potentiation, or inhibition by wortmannin of another intermediate of the alpha-adrenergic signaling cascade. The stimulating effects of both the alpha- and the beta-adrenergic pathways on glucose uptake are independent of changes in cardiac performance. (+info)
(4/1305) Sympathetic nervous system activity and alpha-adrenergic responsiveness in older hypertensive humans.
We have previously demonstrated in normotensive humans an age-associated increase in sympathetic nervous system (SNS) activity combined with appropriate downregulation of alpha-adrenergic responsiveness. Impaired downregulation of alpha-adrenergic responsiveness, despite a comparable level of SNS activity, could contribute to higher blood pressure in older hypertensive humans. We measured arterial plasma norepinephrine (NE) levels and the extravascular NE release rate (NE2) derived from [3H]NE kinetics (to assess systemic SNS activity), and platelet and forearm arterial adrenergic responsiveness in 20 normotensive (N) and in 24 hypertensive (H), otherwise healthy, older subjects (60-75 yr). Although plasma NE levels were similar (N 357 +/- 27 vs. H 322 +/- 22 pg/ml; P = 0.37), NE2 tended to be greater in the hypertensive group (H 2.23 +/- 0.21 vs. N 1.64 +/- 0.20 microgram. min-1. m-2; P = 0. 11), and the NE metabolic clearance rate was greater (H 1,100 +/- 30 vs. N 900 +/- 50 ml/m2; P = 0.004). In the hypertensive group, there was a greater alpha-agonist-mediated inhibition of platelet membrane adenylyl cyclase activity and a NE- but not ANG II-mediated decrease in forearm blood flow. Compared with normotensive subjects, in older hypertensive subjects 1) NE metabolic clearance rate is increased, 2) systemic SNS activity tends to be increased, and 3) arterial and platelet alpha-adrenergic responsiveness is enhanced. These results suggest that heightened SNS activity coupled with enhanced alpha-adrenergic responsiveness may contribute to elevated blood pressure in older hypertensive humans. (+info)
(5/1305) Hemodynamic basis for the acute cardiac effects of troglitazone in isolated perfused rat hearts.
Troglitazone is a thiazolidinedione used for the treatment of NIDDM and potentially for other insulin-resistant disease states. Troglitazone has recently been shown to increase cardiac output and stroke volume in human subjects. These actions are thought to be mediated by the reduction of peripheral resistance, but a potential direct effect on cardiac function has not been studied. Therefore, we investigated the direct cardiac hemodynamic effects of troglitazone in isolated perfused rat hearts. Five groups of hearts were studied. Hearts were tested under isovolumetric contraction with a constant coronary flow, and troglitazone (0.2, 0.5, and 1.0 micromol) was administered by bolus injection. Peak isovolumetric left ventricular pressure (LVPmax), peak rate of rise of LVP (dP/dt(max)), and peak rate of fall of LVP (dP/dt(min)) were significantly increased 1 min after troglitazone administration in a dose-dependent manner, while the heart rate (HR) and coronary perfusion pressure (CPP) were significantly decreased (P < 0.05). HR was then fixed by pacing and/or CPP was fixed with nitroprusside to eliminate any effect of the two variables on the action of troglitazone. With constant HR and/or constant CPP, the effect of troglitazone on LVPmax, dP/dt(max), and dP/dt(min) was still unchanged. In addition, the positive inotropic, positive lusitropic, and negative chronotropic actions of troglitazone were not influenced even when hearts were pretreated with prazosin, propranolol, or nifedipine. In conclusion, troglitazone has direct positive inotropic, positive lusitropic, negative chronotropic, and coronary artery dilating effects. The inotropic and chronotropic actions of troglitazone are not mediated via adrenergic receptors or calcium channels. These findings have important clinical implications for diabetic patients with congestive heart failure. (+info)
(6/1305) Non-specific action of methoxamine on Ito, and the cloned channels hKv 1.5 and Kv 4.2.
The alpha1-adrenoceptor agonist methoxamine acted independently of receptor activation to reduce Ito and the sustained outward current in rat ventricular myocytes, and hKv 1.5 and Kv 4.2 cloned K+ channel currents. Two hundred microM methoxamine reduced Ito by 36% in the presence of 2 microM prazosin, and by 37 and 38% after preincubation of myocytes with either N-ethylmaleimide or phenoxybenzamine (n=6). The EC50 values at +60 mV for direct reduction of Ito, hKv 1.5, and Kv 4.2 by methoxamine were 239, 276, and 363 microM, respectively, with Hill coefficients of 0.87-1.5. Methoxamine accelerated Ito and Kv 4.2 current inactivation in a concentration- and voltage-dependent manner. Apparent rate constants for methoxamine binding and unbinding gave Kd values in agreement with EC50 values measured from dose-response relations. The voltage-dependence of block supported charged methoxamine binding to a putative intracellular site that sensed approximately 20% of the transmembrane electrical field. In the presence of methoxamine, deactivating Kv 4.2 tail currents displayed a distinct rising phase, and were slowed relative to control, such that tail current crossover was observed. These observations support a dominant mechanism of open channel block, although closed channel block could not be ruled out. Single-channel data from hKv 1.5 patches revealed increased closed times with blank sweeps and decreased burst duration in the presence of drug, and a reduction of mean channel open time from 1.8 ms in control to 0.4 ms in 500 microM methoxamine. For this channel, therefore, both open and closed channel block appeared to be important mechanisms for the action of methoxamine. (+info)
(7/1305) Human dynamic closed-loop accommodation augmented by sympathetic inhibition.
PURPOSE: A ciliary alpha-adrenoceptor accommodative effect has been proposed, caused by a small population of alpha1-inhibitory receptors in excised human ciliary muscle. This study was intended to investigate the effect on the closed-loop dynamic accommodative process of modulating alpha1-adrenoceptor activity by topical instillation of the alpha1-adrenergic agonist, phenylephrine hydrochloride. METHODS: A group of 10 visually normal subjects viewed a photopic (30 candela/m2) high-contrast Maltese cross, which was modulated sinusoidally (0.05-0.6Hz) and stepwise over a 2-D range (2-4 D). Monocular temporal accommodation responses were measured using a continuously recording dynamic tracking infrared optometer under two trial conditions: after instillation of saline control solution and 50 minutes subsequent to the instillation of 0.27 microl 0.4% benoxinate hydrochloride and 0.27 microl 2.5% phenylephrine hydrochloride. Pupil size and accommodative amplitude were measured at 90-second intervals for 50 minutes after drug instillation. All accommodative measurements were recorded through a fixed 4-mm pupil. RESULTS: A significant reduction in accommodative amplitude (11%; P < 0.05) was recorded, whereas pupil size showed a significant increase (33%; P < 0.05). No significant change in step-response dynamics was observed. However, phenylephrine hydrochloride caused a significant increase in accommodative gain in the low and midtemporal frequency ranges compared with the effect of a saline control treatment. No significant variation in phase lag was observed. CONCLUSIONS: For the first time in humans, this study shows that augmentation of the alpha1-inhibitory sympathetic contribution results in increased accommodative gain at low and midtemporal frequencies, which is consistent with findings in animal studies. (+info)
(8/1305) Receptor subtype mediating the adrenergic sensitivity of pain behavior and ectopic discharges in neuropathic Lewis rats.
Receptor subtype mediating the adrenergic sensitivity of pain behavior and ectopic discharges in neuropathic Lewis rats. We attempted to identify the subtype of alpha-adrenergic receptor (alpha-AR) that is responsible for the sympathetic (adrenergic) dependency of neuropathic pain in the segmental spinal injury (SSI) model in the Lewis strain of rat. This model was chosen because our previous study showed that pain behaviors in this condition are particularly sensitive to systemic injection of phentolamine (PTL), a general alpha-AR blocker. We examined the effects of specific alpha1- and alpha2-AR blockers on 1) behavioral signs of mechanical allodynia, 2) ectopic discharges recorded in the in vivo condition, and 3) ectopic discharges recorded in an in vitro setup. One week after tight ligation of the L5 and L6 spinal nerves, mechanical thresholds of the paw for foot withdrawals were drastically lowered; we interpreted this change as a sign of mechanical allodynia. Signs of mechanical allodynia were significantly relieved by a systemic injection of PTL (a mixed alpha1- and alpha2-AR antagonist) or terazosin (TRZ, an alpha1-AR antagonist) but not by various alpha2-AR antagonists (idazoxan, rauwolscine, or yohimbine), suggesting that the alpha1-AR is in part the mediator of the signs of mechanical allodynia. Ongoing ectopic discharges were recorded from injured afferents in fascicles of the L5 dorsal root of the neuropathic rat with an in vivo recording setup. Ongoing discharge rate was significantly reduced after intraperitoneal injection of PTL or TRZ but not by idazoxan. In addition, by using an in vitro recording setup, spontaneous activity was recorded from teased dorsal root fibers in a segment in which the spinal nerve was previously ligated. Application of epinephrine to the perfusion bath enhanced ongoing discharges. This evoked activity was blocked by pretreatment with TRZ but not with idazoxan. This study demonstrated that both behavioral signs of mechanical allodynia and ectopic discharges of injured afferents in the Lewis neuropathic rat are in part mediated by mechanisms involving alpha1-ARs. These results suggest that the sympathetic dependency of neuropathic pain in the Lewis strain of the rat is mediated by the alpha1 subtype of AR. (+info)