Emerging evidence suggests that the intravenous injection of bone marrow-derived stromal cells (BMSC) improves renal function after acute tubular injury, but the mechanism of this effect is ...controversial. In this article, we confirm that intravenous infusion of male BMSC reduced the severity of cisplatin-induced acute renal failure in adult female mice. This effect was also seen when BMSC (or adipocyte-derived stromal cells (AdSC)), were given by intraperitoneal injection. Infusion of BMSC enhanced tubular cell proliferation after injury and decreased tubular cell apoptosis. Using the Y chromosome as a marker of donor stromal cells, examination of multiple kidney sections at one or four days after cell infusion failed to reveal any examples of stromal cells within the tubules, and only rare examples of stromal cells within the renal interstitium. Furthermore, conditioned media from cultured stromal cells induced migration and proliferation of kidney-derived epithelial cells and significantly diminished cisplatin-induced proximal tubule cell death in vitro. Intraperitoneal administration of this conditioned medium to mice injected with cisplatin diminished tubular cell apoptosis, increased survival, and limited renal injury. Thus, marrow stromal cells protect the kidney from toxic injury by secreting factors that limit apoptosis and enhance proliferation of the endogenous tubular cells, suggesting that transplantation of the cells themselves is not necessary. Identification of the stromal cell-derived protective factors may provide new therapeutic options to explore in humans with acute kidney injury.
We examined reserpine‐induced chemical denervation supersensitivity with special reference to alpha‐1 adrenoceptor (AR) subtypes.
Chronic treatment with reserpine for 2 weeks depleted noradrenaline ...in the tail artery and spleen of rats. Noradrenaline in the thoracic aorta was negligible before and after reserpine treatment.
The treatment with reserpine produced supersensitivity in the contractile responses of the rat tail artery to phenylephrine, 5‐HT and KCl, resulting in leftward shift of concentration–response curves (11.6‐, 2.5‐ and 1.1‐fold at EC50 value, respectively). These results suggest a predominant sensitization of the alpha‐1 AR‐mediated response by reserpine treatment.
BMY 7378 at a concentration (30 nM) specific for blocking the alpha‐1D AR subtype, but not KMD‐3213 at a concentration (10 nM) selective for blocking the alpha‐1A AR subtype, inhibited the supersensitivity of the phenylephrine‐induced response in the reserpine‐treated artery. On the other hand, the response to phenylephrine in reserpine‐untreated artery was selectively inhibited by the same concentration of KMD‐3213, but not by BMY 7378. Prazosin, a subtype‐nonselective antagonist, blocked the responses to phenylephrine with the same potency, regardless of reserpine treatment.
In the thoracic aorta and spleen, no supersensitivity was produced in the responses to phenylephrine by reserpine treatment.
In a tissue segment‐binding study using 3H‐prazosin, the total density and affinity of alpha‐1 ARs in the rat tail artery were not changed by treatment with reserpine. However, alpha‐1D AR with high affinity for BMY 7378 was significantly detected in reserpine‐treated tail artery, in contrast to untreated artery. Decreases in alpha‐1A AR with high affinity for KMD‐3213 and alpha‐1B AR with low affinities for KMD‐3213 and BMY 7378 were also estimated in reserpine‐treated tail artery.
Alpha‐1D AR mRNA in rat tail artery increased to three‐folds by reserpine treatment, whereas the levels of alpha‐1A and 1B mRNAs were not significantly changed.
The present results suggest that chronic treatment with reserpine affects the expression of alpha‐1 AR subtypes of rat tail artery and that the induction of alpha‐1D ARs with high affinity for catecholamines is in part associated with reserpine‐induced supersensitivity.
British Journal of Pharmacology (2004) 142, 647–656. doi:10.1038/sj.bjp.0705817
Serotonin (5-HT(2)) antagonists show high affinity for the alpha(1)-adrenoceptor (alpha(1)-AR) in addition to the 5-HT(2) receptor. In the present study we compared the pharmacological ...characteristics of a new 5-HT(2) antagonist sarpogrelate and its active metabolite M-1 with those of ketanserin on human recombinant alpha(1)-AR subtypes. In the binding study, sarpogrelate, M-1 and ketanserin produced concentration-dependent inhibition of (3)H-prazosin binding to alpha(1)-ARs. Among the three drugs, ketanserin showed the highest affinity for alpha(1a)-, alpha(1b)- and alpha(1d)-ARs (pKi 8.0, 8.3 and 7.6, respectively). Sarpogrelate had a relatively low affinity for the three subtypes (6.3, 6.4 and 6.3, respectively) and M-1 showed medium affinity (7.1, 7.1 and 6.1, respectively). Chinese hamster ovary (CHO) cells expressing each alpha(1)-AR subtype showed concentration-dependent inositol phosphate (IP) accumulation in response to phenylephrine. The concentration response curves were shifted to the right by three drugs, and the pKb values were close to the pKi values in the binding study. In addition to these effects, sarpogrelate and M-1, but not ketanserin produced an increase in the basal IP level of alpha(1d)-expressed CHO cells, although the increase was less than that of phenylephrine. The present results indicate that sarpogrelate and M-1 have antagonistic activity to the three alpha(1)-AR subtypes, but their affinities are significantly lower than those of ketanserin.
We established three human embryonic kidney (HEK) 293 cell lines stably expressing alpha(1)-adrenoceptor (AR) subtypes, one (alpha(1A), (1B)-AR) coexpressing both receptors and the other two ...(alpha(1A)-AR and alpha(1B)-AR) expressing each receptor in isolation. In the alpha(1A), (1B)-AR cells, both receptors were clearly distinguished by the alpha(1A)-selective ligands (-)-1(3-hydroxypropyl)-5-((2R)-2-(2-(2,2,2-trifluoroethyloxyphenyl)oxy)ethylamino)propyl)-2,3-dihydro-1H-indole-7-carboxamide (KMD-3213) and methoxamine, but not by the subtype-nonselective ligands prazosin and phenylephrine. In all three cell lines, phenylephrine caused a concentration-dependent increase in inositol phosphates and an increase in extracellular signal-regulated kinase 1/2 (ERK1/2) activation. However, there was a 2-fold or greater maximal response to phenylephrine and a somewhat higher agonist potency in ERK1/2 activation in the alpha(1A,1B)-AR cells, compared with the responses of cells expressing either receptor individually (alpha(1A)-AR or alpha(1B)-AR). Furthermore, the antagonistic affinities of prazosin (pK(b) of 10.1) and KMD-3213 (9.4) for inhibiting the phenylephrine response were intermediate between the values for inhibition in alpha(1A)-AR cells (prazosin, 9.3; KMD-3213, 10.5) and alpha(1B)-AR cells (prazosin, 11.0; KMD-3213, 8.1). The inhibitor pK(b) values in alpha(1A), (1B)-AR also differed from their ligand binding affinities measured in alpha(1A)-AR and alpha(1B)-AR cells. In contrast, the alpha(1A)-selective agonist methoxamine, which did not activate alpha(1B)-AR cells, stimulated either alpha(1A,) (1B)-AR or alpha(1A)-AR cells with a comparable potency and maximum effectiveness. Our data indicate that when coexpressed in the same cell, the activation of common pathways by individual AR receptor subtypes by a nonselective agonist can exhibit enhanced responsiveness and a distinct antagonist affinity compared with the parameters for the same receptors, when expressed alone in the same cell background.
We established three HEK cell lines stably expressing α1-adrenoceptor (AR) subtypes, one (α1a, 1b-AR) co-expressing both receptors and the other two (α1a-AR; α1b-AR) expressing each receptor in ...isolation. In all three cell lines, phenylephrine caused a concentration-dependent increase in inositol phosphates accumulation and ERK activation. However, there was a greater maximal response to phenylephrine and a higher agonist potency in the α1a, 1b-AR cells, compared with the responses of cells expressing either receptor individually. The antagonistic affinities of prazosin and KMD-3213 for inhibiting the phenylephrine response were intermediate between the values for inhibition in α1a-AR cells and α1b-AR cells. The pKb values in α1a, 1b-AR also differed from their binding affinities measured in α1a-AR and α1b-AR cells. Thus, the co-activation of co-expressed α1a, 1b-AR subtypes exhibits enhanced responsiveness and a distinct antagonist affinity compared with the parameters for the same receptors, when expressed alone.
Alpha-1 adrenoceptors (ARs) are members of the G protein-coupled receptors and play critical roles in the regulation of a variety of physiological processes. Recently, recombinant alpha-id subtype ...was shown to have a relatively high constitutive activity in the absence of agonist. In the present study, we examined the effects of various drugs on the constitutive activity. At first, CHO cells expressed alpha-1d AR was treated for 72 hours with 22 drugs and the effects on receptor density was examined. The most drugs including BMY7378, tamsulosin and WB4101 produced a significant increase in alpha-1d AR. However, HEAT and prazosin failed to change the density and inhibited the upregulation induced by BMY7378. Basal production of inositol phosphates was reduced by BMY7378 and other drugs in CHO cells expressed alpha- 1d AR but in mock cells. However, HEAT and prazosin inhibited the effect of BMY7378 on inositol phosphate production without affecting the basal production. These results indicate that most drugs including BMY7378 are inverse agonists at alpha-1d AR, but that HEAT and prazosin behave like neutral antagonists, which have little effect on the constitutive activity.
Radioligand binding assay is one of the most useful tool to characterize many receptors. Most of binding studies have been done with membrane fractions prepared from tissues. However, there are some ...limitations in this method, such as limited amount of tissue and low yield due to fractionation. In this study, we developed a tissue segment binding method, which allows detecting native pharmacological profiles in small tissues. Rat tail artery and thoracic aorta were cut into small segments and incubated with 3H-prazosin at 4゜ C for 12 hours. Then, tissue segments were washed with buffer and dissolved in NaOH solution for measurement of the radioactivity and protein. One set of saturation or displacement experiment was carried out with the use of segments of tail artery or thoracic aorta isolated from one rat. In saturation experiments, total α1-adrenoceptor density of tail artery and thoracic aorta were 546 fmol/mg protein and 138 fmol/mg protein, respectively. Displacement experiments with KMD-3213 and BMY-7378 showed coexistence of α1A and α1B-adrenoceptors in rat tail artery, and α1B-and α1D-adrenoceptors in rat thoracic aorta. These results were compared with those obtained in membrane binding study and functional study, suggesting that the developed tissue segment binding method is very valuable one to evaluate the receptors in small tissues.
Supersensitivity is one of classical and pharmacological phenomena, and may be caused by a variety of procedures, including surgical and chemical denervation and chronic treatment with antagonists. ...We examined reserpine-induced chemical denervation supersensitivity with special reference to arterial alpha 1-adrenoceptor (alpha 1-AR) subtypes. Chronic treatment with reserpine for 2 weeks produced supersensitivity in the contractile response to phenylephrine of isolated rat tail artery, resulting a leftward shift of concentration-response curve (10 fold shift at EC50 value). This supersensitivity in reserpine-treated artery was selectively inhibited by BMY7378 (alpha 1D-AR selective antagonist) but not by KMD-3213 (alpha 1A-AR selective antagonist). On the other hand, the response to phenylephrine in reserpine-untreated artery was inhibited by KMD-3213 but not by BMY7378. Tissue segment binding study with 3H-prazosin revealed that high affinity binding sites for BMY7378 were detected with a proportion of 32% in reserpine-treated tail artery but absent in untreated artery, although total density of alpha 1-ARs was not changed by reserpine-treatment. The present results strongly suggest that reserpine-induced supersensitivity of rat tail artery is caused by a selective induction of alpha 1D-AR subtype.
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