Agonist-specific regulation of the delta-opioid receptor

Delta opioid receptor (DOR) agonists are attractive potential analgesics, since these compounds exhibit strong antinociceptive activity with relatively few side effects. In the past decade, several novel classes of delta-opioid agonists have been synthesized. Recent experimental data indicate that structurally distinct opioid agonists interact differently with the delta-opioid receptor. Consequently, individual agonist-bound DOR conformations may interact differently with intracellular proteins. In the present paper, after a brief review of the cellular processes that contribute to homologous desensitization of the DOR signaling, we shall focus on experimental data demonstrating that chemically different agonists differ in their ability to phosphorylate, internalize, and/or down-regulate the DOR. Homologous regulation of the opioid receptor signaling is thought to play an important role in the development of opioid tolerance. Therefore, agonist-specific differences in DOR regulation suggest that by further chemical modification, delta-selective opioid analgesics can be designed that exhibit a reduced propensity for analgesic tolerance.     

A unique regulatory profile and regional distribution of [3H]pirenzepine binding in the rat provide evidence for distinct M1 and M2 muscarinic receptor subtypes

We recently demonstrated that the non-classical muscarinic receptor antagonist [³H]pirenzepine ([³H]PZ) identifies a high affinity population of muscarinic sites in the rat cerebral cortex. We now report that cortical muscarinic sites to which [³H]PZ binds with high affinity are modulated by ions but not guanine nucleotides. We also have examined equilibrium [³H]PZ binding in homogenates of various rat tissues using a new rapid filtration assay. All regional saturation isotherms yielded a similar high affinity dissociation constant (K_d = 2 ? 8 nM) in 10 mM sodium-potassium phosphate buffer. Receptor density (B_max in fmol/mg tissue) varied as follows: corpus striatum = 154.5, cerebral cortex = 94.6, hippocampus = 94.3, ileum = 1.3, cerebellum = 1.0, and heart = 0.45. The cerebral cortex and hippocampus possess 61 percent of striatal binding sites, while the ileum, cerebellum and heart contain only 0.84 percent, 0.65 percent and 0.29 percent of striatal sites respectively. The [³H]PZ sites in heart, ileum, and cerebellum represent 3.1 percent, 9.6 percent, and 10.4 percent of the sites obtained by using [³H](?)quinuclidinyl benzilate. Thus, [³H]PZ labels high affinity muscarinic receptor binding sites with a tissue distribution compatible with the concept of distinct M₁ and M₂ receptor subtypes. Accordingly, regions such as heart, cerebellum, and ileum would be termed M₂, though each have an extremely small population of the M₁ high affinity [³H]PZ site. [³H]PZ therefore appears to be a useful ligand for M₁ receptor identification. Furthermore, the inability to demonstrate a significant effect of guanine nucleotides upon high affinity [³H]PZ binding to putative M₁ receptors suggests that M₁ sites may be independent of a guanine regulatory protein.     

The measurement of free nitrendipine in human serum by an equilibrium dialysis - radioreceptor assay

A radioreceptor assay using [³H]nitrendipine and rat cerebral cortical membranes, in conjunction with equilibrium dialysis, measures the unbound (free) level of nitrendipine in human sera. The sensitivity of the assay is 0.1–0.2 picomoles/ml and is linear from 4 × 10^?11 to 4 × 10^?9 M nitrendipine. Other dihydropyridine calcium channel antagonists may be measured using this assay if these compounds are used to generate the standard curve. Blank serum interferes with specific [³H]nitrendipine binding (24 percent inhibition per 20 μ1 serum) whereas serum dialysates do not. Total serum nitrendipine levels may be measured, but the sensitivity of the assay is decreased due to interference by serum. Nitrendipine is highly protein bound in serum (93 – 99 percent). This protein binding is essentially unchanged over a serum concentration from 1 to 100 ng/ml. This assay is suitable for pharmacokinetic and pharmacodynamic studies.     

The binding of [3H]nitrendipine to receptors for calcium channel antagonists in the heart,cerebral cortex,and ileum of rats

The binding properties of the calcium channel antagonist, [³H]nitrendipine, were investigated in homogenates of the rat cerebral cortex, heart and ileum. The specific component of [³H]nitrendipine binding was consistent with mass-action behavior and was characterized by a high affinity dissociation constant in the range of 0.1 ? 0.3 nM. A variety of other calcium channel antagonists inhibited the binding of [³H]nitrendipine with K_i's that agree generally with the ability of these drugs to block contractions of cardiac and smooth muscle. The inhibition of [³H]nitredipine binding by other dihydropyridines was consistent with competitive antagonism whereas the inhibition caused by verapamil and D600 resembled negative heterotropic cooperativity. Consistent with this latter postulate was the observation that the kinetics of [³H]nitrendipine binding are altered by verapamil, with both the association rate and the dissociation rate being increased. La⁺³ and several divalent cations caused an inhibition of [³H]nitrendipine with the rank order of potency being Cd⁺² > La⁺³ > Ni⁺² > Co⁺² ? Mn⁺² > Mg⁺² ? Ba⁺² > Ca⁺².     

Characterization of postsynaptic β-adrenergic receptors and presynaptic muscarinic cholinergic receptors in the rat spleen

The β-adrenergic and muscarinic cholinergic receptors in the splenic homogenates of control and 6-hydroxydopamine (6-OHDA) treated rats were characterized. The specific binding of [³H]dihydroalprenolol (DHA) and [³H]quinuclidinyl benzilate (QNB) in the rat spleen were saturable and of high affinity and showed pharmacological specificity of splenic β-adrenergic and muscarinic cholinergic receptors. Following 6-OHDA treatment, the Bmax value for specific [³H](-)DHA binding to the rat spleen was significantly increased by 26 percent and 22 percent compared to control at 2 and 3 weeks without a change in the Kd. In contrast, there was a 38 percent decrease in the Bmax for [³H](-)QNB in the 6-OHDA treated rat spleen at 2 and 3 weeks respectively without a change in the Kd. The Bmax value at 5 weeks was significantly greater than that at 2 or 3 weeks. The splenic norepinephrine (NE) concentration was markedly reduced by the 6-OHDA treatment at 1 to 3 weeks, while there was a significant recovery in the splenic NE concentration at 5 weeks. Thus, our results strongly suggest that we are biochemically localizing muscarinic cholinergic receptors on the sympathetic nerves of the rat spleen and that the β-adrenergic receptors of the spleen are localized postsynaptically.     

High affinity renal [3H]flunitrazepam binding: Characterization,localization, and alteration in hypertension

The binding of [³H]flunitrazepam was studied in membranes prepared from the kidney and cerebral cortex of unilaterally nephrectomized rats made hypertensive by simultaneous deoxycorticosterone acetate (DOCA) and NaCl administration. A significant 35–43% increase in the number of [³H]flunitrazepam binding sites (Bmax) was found in the renal membranes prepared from the hypertensive rats; there was no change in the density of binding sites in the membranes obtained from the cerebral cortex. The K_d of [³H]flunitrazepam binding did not change either in the renal or in the cerebral membranes (～ 12 nM in the kidney and ～2.0 nM in the brain). Drug specificity studies with renal membranes showed that the inhibition of [³H]flunitrazepam binding by various benzodiazepines did not jibe with their pharmacologic potency as anxiolytic agents. An intrarenal distribution of specific [³H]flunitrazepam binding was found in the bovine kidney; specific binding was greatest in the outer cortex and virtually absent in the medulla, the minor calyx and the renal artery. The evidence that the renal benzodiazepine binding site is of high affinity, is specific, has a unique distribution, and is regulated during hypertension suggests that it may be associated with an important pathophysiologic structure.     

Cloning of the rat M3, M4 and M5 muscarinic acetylcholine receptor genes by the polymerase chain reaction (PCR) and the pharmacological characterization of the expressed genes.

The coding sequence of the rat m3, m4 and m5 subtypes of muscarinic acetylcholine receptor (mAChR) genes was amplified by the polymerase chain reaction (PCR), cloned, and expressed in the murine fibroblast (B82) cell line. Sequencing of the cloned genes revealed some nucleotide differences when compared with the DNA sequence published in the literature. When the different sequence appeared in only one clone obtained by PCR, it was considered an error of the polymerase. The overall error frequency in the 25 cycles of PCR with either Taq polymerase or Replinase was 1 nucleotide in 1,692 base pairs. In order to evaluate the different nucleotide sequence from a PCR product as an error or as an allelic variant, at least three different clones were sequenced. The cloned genes were each stably expressed in a B82 cell line and pharmacologically evaluated. The affinity of the different antagonists to the muscarinic receptor subtypes was determined by [3H](-)MQNB/ligand inhibition experiments. In the m3, m4 and m5 transfected cells, carbachol appeared to stimulate [3H]inositol monophosphate (IP1) accumulation. Carbachol, at 3 microM, appeared to suppress the forskolin-stimulated cAMP formation in the m4 transfected cells. These findings suggest these mAChRs amplified by PCR, cloned, and expressed in the B82 cell lines exhibit the pharmacological characteristics of the muscarinic receptor subtypes.     

Synthesis and self-alkylation of isotope-coded affinity tag reagents

A pair of ICAT reagents, N-(13-iodoacetamido-2,2,3,3,11,11,12,12-octadeutero-4,7,10-trioxa-tridecanyl)biotinamide (8d, ICAT-d(8)) and N-(13-iodoacetamido-4,7,10-trioxa-tridecanyl)biotinamide (8c, ICAT-d(0)), and an alternative pair of ICAT reagents, N-(10-iodoacetamido-2,5,5,6,6,9-hexadeutero-4,7-dioxa-decanyl)biotinamide (8b, s-ICAT-d(6)) and N-(10-iodoacetamido-4,7-dioxa-decanyl)biotinamide (8a, s-ICAT-d(0)), were successfully synthesized. A mixture of sodium borohydride and cobalt(II) chloride reduced the intermediate dinitrile to the diamine without loss of the deuterium labels, which occurred when Raney nickel was the reducing agent. The problem caused by unsymmetrical biotinylation of the intermediate diamine was solved by using the solid-phase method in which one end of the diamine was attached to a chlorotrityl chloride resin, followed by biotinylation of the resin-bound amine. The self-alkylation of ICAT reagents that accounted for their instability and their limitations in the applications was also studied.     

Hip joint replacement surgery for idiopathic osteoarthritis aggregates in families

In order to determine whether there is a genetic component to hip or knee joint failure due to idiopathic osteoarthritis (OA), we invited patients (probands) undergoing hip or knee arthroplasty for management of idiopathic OA to provide detailed family histories regarding the prevalence of idiopathic OA requiring joint replacement in their siblings. We also invited their spouses to provide detailed family histories about their siblings to serve as a control group. In the probands, we confirmed the diagnosis of idiopathic OA using American College of Rheumatology criteria. The cohorts included the siblings of 635 probands undergoing total hip replacement, the siblings of 486 probands undergoing total knee replacement, and the siblings of 787 spouses. We compared the prevalence of arthroplasty for idiopathic OA among the siblings of the probands with that among the siblings of the spouses, and we used logistic regression to identify independent risk factors for hip and knee arthroplasty in the siblings. Familial aggregation for hip arthroplasty, but not for knee arthroplasty, was observed after controlling for age and sex, suggesting a genetic contribution to end-stage hip OA but not to end-stage knee OA. We conclude that attempts to identify genes that predispose to idiopathic OA resulting in joint failure are more likely to be successful in patients with hip OA than in those with knee OA.