Charlene D. Richardson

Subtype Specific Regulation of Human Vascular α1-Adrenergic Receptors by Vessel Bed and Age

Background—α1-adrenergic receptors (α1ARs) regulate blood pressure, regional vascular resistance, and venous capacitance; the exact subtype (α1a, α1b, α1 d) mediating these effects is unknown and varies with species studied. In order to understand mechanisms underlying cardiovascular responses to acute stress and chronic catecholamine exposure (as seen with aging), we tested two hypotheses: (1) human α1AR subtype expression differs with vascular bed, and (2) age influences human vascular α1AR subtype expression. Methods and Results—Five hundred vessels from 384 patients were examined for α1AR subtype distribution at mRNA and protein levels (RNase protection assays, ligand binding, contraction assays). Overall vessel α1AR density is 16±2.3fmol/mg total protein. α1aAR predominates in arteries at mRNA (P<0.001) and protein (P<0.05) levels; all 3 subtypes are present in veins. Furthermore, α1AR mRNA subtype expression varies with vessel bed (α1a higher in splanchnic versus central arteries, P<0.05); competiti...

alpha1-ADRENERGIC RECEPTOR SUBTYPES IN HUMAN DETRUSOR

PURPOSE To identify and quantitate alpha1-adrenergic receptor (alpha1AR) subtype expression in human detrusor. MATERIALS AND METHODS Initial studies to determine alpha1AR expression in human detrusor were performed using saturation binding with [125I]HEAT. Once the presence of alpha1ARs was documented, subtype (alpha1a, alpha1b, alpha1d) expression at the mRNA level (and comparison with rat) was determined with RNase protection assays (human detrusor) and RT-PCR (human detrusor, rat whole bladder). Competition binding analysis with the alpha1dAR-selective ligand BMY7378 was used to measure alpha1AR subtype expression at a protein level in human detrusor. RESULTS Alpha1AR expression in human detrusor was low but reproducible (6.3 +/- 1.0 fmol./mg. total protein). RNase protection assays performed on total RNA extracted from human detrusor revealed the following alpha1AR subtype expression: alpha1d (66%) > alpha1a (34%), and no alpha1b. RT-PCR confirmed alpha1AR subtype mRNA distribution in human detrusor with alpha1d (approximately 60-70%) > alpha1a (approximately 30-40%), and a lack of alpha1b mRNA. Rat whole bladder expressed different alpha1AR subtype mRNA than human detrusor, with alpha1a approximately alpha1b approximately alpha1d. The presence of alpha1d > alpha1a expression in human detrusor was confirmed at a protein level by competition analysis utilizing BMY7378 which revealed a two-site fit, with Ki and high affinity binding (66%) consistent with the alpha1dAR subtype. CONCLUSIONS Human detrusor contained two alpha1AR subtypes (alpha1d > alpha1a), a finding that is different from rat, another commonly used animal model. Since non-subtype selective alpha1AR antagonists ameliorate irritative bladder symptoms (in men and women with/without outlet obstruction), and Rec 15/2739 (alpha1a selective antagonist) does not improve symptom scores in BPH, our findings suggest bladder alpha1dARs may provide a potentially novel mechanism underlying these therapeutic benefits.

Pharmacology of tamsulosin: Saturation-binding isotherms and competition analysis using cloned α1-adrenergic receptor subtypes

α1‐adrenergic receptors (α1 ARs) are important in the dynamic component of benign prostatic hyperplasia (BPH). Currently, several α1AR antagonists are being used in the treatment of BPH.

α2-Adrenergic Receptors in Human Dorsal Root Ganglia: Predominance of α2b and α2c Subtype mRNAs

Background:Nonselective α2-adrenergic receptor (α2AR) agonists (e.g., clonidine) mediate antinociception in part through α2ARs in spinal cord dorsal horn; however, use of these agents for analgesia in humans is limited by unwanted sedation and hypotension. The authors previously demonstrated α2a ≈ α

In situ hybridization: identification of rare mRNAs in human tissues

In situ hybridization is used for detection of RNA expression when conservation of tissue architecture is important. Most in situ hybridization protocols are written for tissues from animals (i.e., rat) which can be harvested and preserved rapidly. In contrast, human tissue is more difficult to obtain, hence in situ hybridization experiments must frequently be performed with less than optimal tissue preservation. This procedure details hybridization of a radiolabeled single-stranded RNA probe (riboprobe) to complementary sequences of cellular RNA in human tissue sections. This method enables detection of rare mRNA species in specific cell types of human tissue, offering distinct advantages over other in situ methods due to increased sensitivity. In particular, we have found that UV cross-linking and ribonuclease treatment protocols need to be altered for human tissues to ensure successful results, making this protocol unique to those previously described. In situ hybridization experiments can be performed using either DNA or RNA probes. RNA probes are advantageous since they form stable hybrids, are single-stranded, have little or no reannealing during hybridization, and can be synthesized to high specific activity. RNA probes can be readily created utilizing SP6, T3, or T7 promoters in both sense and antisense orientations to provide non-specific (control) and specific probes. Disadvantages of RNA riboprobes include a tendency for RNA to stick non-selectively more than DNA, and degradation by RNase (hence strict adherence to RNase-free precautions is mandatory during most of the protocol). The following protocol includes: (1) preparation of human tissues (tissue fixation and sectioning are highlighted as critical for probe penetration, preservation of tissue architecture, retention of tissue RNA, and overall success); (2) generation of radiolabeled riboprobes (total incorporation of radionucleotide is important to increase sensitivity; 35S was chosen as a compromise between excellent sensitivity, cellular resolution, and required exposure times (compared with 32P or 3H); non-isotopic methods have not been tested in a side-by-side comparison with 35S in human tissues by us, but theoretically might offer faster exposure times while maintaining high resolution); (3) hybridization conditions (stringency, temperature, washes, tissue dehydration); and (4) sample visualization (application of photographic emulsion, developing, fixing, staining, and counterstaining of individual slides).

Multiple Potential Regulatory Elements in the 5′ Flanking Region of the Human al,-Adrenergic Receptor:Short Communication

In spite of their critical importance in myocardial hypertrophy and benign prostatic hyperplasia, nothing is known about mechanisms underlying transcriptional regulation of αla-adrenergic receptors (αl, ARs). Therefore we cloned 6.2kb of novel sequence upstream of the initiator ATG in the human αlaAR gene. Sequence analysis reveals a TATA-less promoter, the presence of several initiator (Inr) consensus sequences, multiple GC rich regions consistent with Sp-1 binding, and consensus sequences for AP-1 and AP-2 as well as putative cis transcriptional regulatory elements for binding of CREB (cyclic-AMP response element binding protein), glucocorticoids, estrogen, and insulin. Compared to the αlbAR, the αlaAR has several more cis regulatory elements, suggesting more complex regulation. The importance of αlaARs in human disease makes it imperative to determine mechanisms underlying transcription and ultimately expression of this receptor. These studies can now be undertaken with the availability of human αlaAR ...

Hypotension resistant to therapy with α receptor agonists complicating cardiopulmonary bypass: Lithium as a potential cause

L ithium is the primary drug used for the treatment of bipolar (manic-depressive) disorders (1); it is also used for the treatment of other neurologic and psychiatric diseases, including aggressive and self-mutilating behavior (2) and cluster headaches (3). Patients receiving lithium may demonstrate a number of side effects, including tremor, confusion, cardiac conduction disturbances, polyuria, and hypothyroidism. Lithium has several side effects important to the anesthesiologist. It prolongs sleep time induced by barbiturates (4,5), enhances neuromuscular blockade produced by succinylcholine (6) and pancuronium (71, and may produce cardiac conduction abnormalities. Despite its widespread use, very little is known about lithium’s exact mode of action. An important landmark in understanding lithium’s mechanism of action occurred when Allison and Stewart (8) demonstrated that lithium decreases the inositol concentration in rat cerebral cortex. Decreased inositol concentrations in brain were later associated with accumulation of inositol phosphate, resulting from an inhibition of the enzyme inositol-1-phosphatase (9). Hence, lithium may exert therapeutic actions by interfering with the regeneration of phosphoinositides, ubiquitous cellular second messengers; lithium may therefore play an important role in synaptic transmission (10,111. This theory, called the “inositol depletion hypothesis,” offers a plausible explanation for lithium action consistent with known therapeutic and pharmacological characteristics (12,131. The phosphoinositide pathway is a common intracellular second messenger signaling system (14) mediating numerous receptor-stimulated cellular functions. a,-Adrenergic receptors (a,ARs) couple via