A principle tenet in pharmacology is that neurotransmitters, drugs, and hormones interact with specific receptor sites to produce or inhibit a characteristic physiological response. In the case of synaptic transmission and many hormone responsive systems, these specific receptor sites are presumed to be integral membrane proteins. The purification folds obtained by the methods represent a 6500-fold purification from initial detergent extracts and a greater than 80000-fold purification from membrane preparations. As the techniques were found to be extremely successful for the purification of the β2-adrenergic receptor, we sought to purify the β1-adrenergic receptor from the turkey red blood cell. Purification and characterization of hormone and drug receptors has been a difficult task primarily due to the extremely small quantities of these macromolecules in most cell types. β-Adrenergic receptors are closely coupled to the enzyme adenylate cyclase and have been the focus of considerable attention.

Neurotransmitters and drugs interact with specific receptors in order to produce a cellular response. This recognition of specific agonists by receptors is the first step in an amplification process resulting in physiological modulation of homeostasis. The concept of a receptor-mediated mechanism for drug action was first proposed by Langley (1878, 1905). In 1906, examination by Dale of the action of various ergot alkaloid compounds in physiological preparations led to the observation of a number of compounds which would inhibit the excitatory effects of epinephrine without affecting its inhibitory actions. These types of observations provided support for the earliest notions of specific sites for catecholamine receptor interactions. In 1948, Ahlquist categorically demonstrated using a series of several sympathomimetic amines one order of potency in stimulating vasoconstriction and excitation of the uterus and ureters, and a different order of potency for these compounds for stimulation of vasodilation, for inhibition of uterine tone, and for stimulation of the heart. Ahlquist (1948) proposed that these actions of catecholamines were in fact mediated by two distinct populations of receptors which he termed o and 8, and that in many cases, both receptors are present in the same organ or tissue. This proposal of distinct

IBC's 5(th) Annual Conference on Blood Substitutes was held on November 20-21, 1997. Approximately 100 medical researchers, academic scientists, blood substitute company speakers and representatives from major pharmaceutical companies with an interest or partner in the field attended. The papers presented focused on the progress in clinical trials for those compounds in human study, on preclinical models for predicting efficacy in humans, and on novel approaches and agents for the delivery of oxygen. Both perfluorocarbons (PFC) and haemoglobin-based oxygen carriers were described. Several lectures addressed the history of the field and future directions for laboratory and clinical investigation. The following is a summary of some of the presentations. This summary is divided into 3 sections. The first section provides a historical overview and discusses the changes in the perceived need for a blood substitute. Section 2 comprises an update of company activities. The final section focuses on likely future directions for laboratory and human clinical study.

The performance of polyethylene glycol-modified bovine hemoglobin (PEG-Hb) was evaluated in dogs following the replacement of 30% or 50% of their blood volume with PEG-Hb or lactated Ringer's solution (LRS). Dogs fully instrumented with catheters and blood pressure probes were transfused by simultaneous bleeding from the jugular vein and infusion of PEG-Hb or LRS via the cephalic vein. Animals were monitored for abnormal behavior and clinical signs for fourteen days. No mortalities, overt toxicity, changes in body weight, food consumption or ophthalmology, or discernable trends in hematology, blood chemistry coagulation, urinalysis or hemodynamic parameters that could be attributed to PEG-Hb were noted. Blood gas analyses were steady and within physiological ranges. Dose-related histopathological findings of vacuolated histiocytes in the femoral bone marrow, splenic parenchyma, the medulla of the mesenteric and mandibular lymph nodes, and vacuolated sinusoidal cells in the liver and the renal tubular epithelial cells were believed to be related to the phagocytosis and degradation of PEG-Hb by the reticulo-endothelial system. The maintenance of high oxygen levels in the circulation for the two-week treatment period, as well as the insignificant physiological and histopathological findings indicate that PEG-Hb could be a successful blood substitute.

Turkey red blood cell (RBC), Beta/sub 1/-adrenergic receptors (Bar) were prepared to electrophoretic homogeneity and denatured protein used to prepare rabbit anti-Bar antibodies. Anti-Bar activity was confirmed by immuno-adsorption of (/sup 125/I) cyanopindolol (CYP) labeled Bar. The catecholamine biosynthetic enzyme dopamine beta hydroxylase (DBH) was purified from bovine adrenal medullae chromaffin vesicles by ion exchange, size exclusion and concanavalin-A-Sepharose chromatography. Final DBH specific activities were 42 +/- 4 U/mg protein. Homogeneity was confirmed by non-denaturing PAGE. Bar was compared to DBH by anti-Bar antibody cross-reactivity. DBH and Bar were recognized by anti-Bar antibodies on immunoblotting. No interactions were observed with preimmune controls. Similar results were obtained with glycosylated and deglycosylated DBH suggesting that the antibodies recognize DBH amino acid sequence and not associated carbohydrate. Cross-reactive antibodies were purified by affinity chromatography using immobilized DBH and shown to immuno-adsorb (/sup 125/I)CYP labeled Bar. These results suggest that the catecholamine biosynthetic enzyme DBH and Bar may be related in structure.