Structure-activity studies of hydrophobic amino acid replacements at positions 9, 11 and 16 of glucagon

We have designed and synthesized eight compounds 2-9 which incorporate neutral, hydrophobic amino acid residues in positions 9, 11 and 16 of the glucagon molecule: (2) [desHis¹,Va1⁹,11e^11,16] glucagon amide, (3) [desHis¹,Val^9,11,16]glucagon amide, (4) [desHis¹,Va1⁹,Leu^11,16]glucagon amide, (5) [desHis¹,Nle⁹,11e^11,16]glucagon amide, (6) [desHis¹,Nle⁹,Val^11,16]glucagon amide, (7) [desHis¹,Nle⁹,Leu^11,16]glucagon amide, (8) [desHis¹,Val⁹,Leu^11,16,Lys^17,18,Glu²¹]glucagon amide and (9) [desHis¹,Nle⁹,Leu^11,16,Lys^17,18,Glu²¹]glucagon amide. The effect of neutral, hydrophobic residues at positions 9, 11 and 16 led to good binding to the glucagon receptor. Compared to glucagon (IC₅₀= 1.5 nM), analogues 2-9 were found to have IC₅₀ values of 6.0, 6.0, 11.0, 9.0, 2.5, 2.8, 6.5 and 7.0 nM, respectively. When these compounds were tested for their ability to block adenylate cyclase (AC) activity, they were found to be antagonists having no stimulation of adenyl cyclase, with PA₂, values of 6.15, 6.20, 6.30, 7.25, 6.10, 7.30, 6.25 and 7.25, respectively. © Munksgaard 1997.     

Synthetic glucagon antagonists and partial agonists

This paper reports the synthesis and the biological activities of six new glucagon analogues. In these compounds N-terminal modifications of the glucagon sequence were made, in most cases combined with changes in the C-terminal region which had been shown previously to enhance receptor affinity. The design of these analogues was based on [Lys^17.18,Glu²¹]glucagon,¹ a superagonist, which binds five times better than glucagon to the glucagon receptor, and on the potent glucagon antagonist [d -Phe⁴,Tyr⁵,Arg¹²]glucagon, which does not stimulate adenylate cyclase system even at very high concentrations. The N-terminal modifications involved substitution of His¹ by the unnatural conformationally constrained residue, 4,5,6,7-tetrahydro-1H-imidazo[c]pyridine-6-carboxylic acid (Tip) and by desaminohistidine (dHis). In addition we prepared two analogues (6 and 7), in which we deleted the Phe⁶ residue, which was suggested to be part of a hydrophobic patch and involved in receptor binding. The following compounds were synthesized: [Tip¹, Lys^17.18,Glu²¹]glucagon (2); [Tip¹,d -Phe⁴,Tyr⁵,Arg¹²,Lys^17.18,Glu²¹ glucagon (3); [dHis¹,d -Phe⁴,Tyr⁵,Arg¹², Lys^17.18,Glu²¹ glucagon (4); [dHis¹,Asp³,d -Phe⁴,Tyr⁵,Arg¹²,Lys^17.18,Glu²¹]glucagon (5); des-Phe⁶-[Tip¹,D-Phe⁴,Tyr⁵Arg¹²,Glu²¹ glucagon (6); des-Phe⁶-[Asp³,d -Phe⁴,Tyr⁵,Arg¹²,Glu²¹]glucagon (7) The binding potencies of these new analogues relative to glucagon (= 100) are 3.2 (2), 2.9 (3), 10.0 (4), 1.0 (5), 8.5 (6), and 1.7 (7). Analogue 2 is a partial agonist (maximum stimulation of adenylate cyclase (AC) approximately 15% and a potency 8.9% that of glucagon, while the remaining compounds 3-7 are antagonists unable to activate the AC system even at concentrations as high as 10^?5m . In addition, in competition experiments, analogues 3-7 caused a right-shift of the glucagon stimulated adenylate cyclase dose-response curve. Hence these compounds are glucagon receptor antagonists with respect to the glucagon receptor coupled to the adenylate cyclase system.     

EFFECT OF CHRONIC TREATMENT WITH AMLODIPINE IN NON-INSULIN-DEPENDENT DIABETIC RATS

We have investigated the effects of amlodipine on streptozotocin- (STZ) induced neonatal non-insulin-dependent diabetes mellitus (NIDDM) rats. NIDDM was induced by intraperitoneal injection of STZ (70 mg kg⁻¹) to 5-day-old rat pups. The animals were weaned at 30 days and maintained with food and waterad libitumfor 3 months. Amlodipine (5 mg kg⁻¹p.o.) was administered for 6 weeks after the animals were confirmed diabetic (3 months after the STZ injection). A group of control animals were also maintained and this group received citrate buffer 5 days after birth. Fasting- and fed-glucose levels in NIDDM rats were significantly higher than control rats. Treatment with amlodipine reduced the elevated fasting- and fed-glucose levels significantly. Results of the oral glucose tolerance test (OGTT) revealed that glucose tolerance is impaired in the NIDDM rats. There was a marked increase in glucose levels after oral administration of glucose in the control NIDDM rats. Increased glucose levels were found to be associated with increased insulin levels. Treatment with amlodipine in the NIDDM rats caused a decrease in insulin release, however, glucose levels were found to be lowered significantly indicating that amlodipine causes an increase in insulin sensitivity. In conclusion, our data indicated that amlodipine increases insulin sensitivity in neonatal-STZ NIDDM rats.     

Persistent Atrial Standstill—Clinical, Electrophysiological, and Morphological Study

Persistent atrial standstill (PAS) is a rare disorder characterized by absence of atrial activity on the surface and intracavity electrograms, absence of atrial mechanical activity, and inability to electrically stimulate the atria. Four patients (ages 18-60 years) with PAS were evaluated. One of these (no. 3) only had right atrial (RA) standstill, whereas left atrium (LA) showed spontaneous activity and could be stimulated electrically. As RA biopsy is not possible, right ventricular (RV) endomyocardial biopsy (EMB) was obtained to identify possible atrial pathology that revealed inflammatory myocarditis, 2; amyloidosis, 1; and myocardial hypertrophy with fibrosis, 1. Three patients were given permanent pacemakers. One of these with amyloidosis died suddenly. One is lost to follow-up. The others cases are persisting with PAS.     

Evidence for α2-Adrenoceptor Agonist Activity of Minoxidil*

The present investigation was undertaken to study the mechanism of action of minoxidil using various smooth muscle preparations. Minoxidil (4.7 × 10^?6 m to 4.7 × 10^?4 m ) produced a concentration-dependent inhibition of field stimulation-evoked responses in rat anococcygeus muscle and vas deferens. The inhibition produced by minoxidil was antagonized by yohimbine (2.5 × 10^?7 m ). Minoxidil (1.4 × 10^?5 m to 4.7 × 10^?4 m ) also produced a concentration-dependent relaxation in oestrogen-primed potassium chloride-depolarized rat uterus. These responses were blocked not only by yohimbine but also by glibenclamide (202 × 10^?8 m ). Our results suggest that minoxidil possesses α₂-adrenoceptor agonist activity in addition to potassium-channel-opening activity.     

Umbilical Arterial Doppler Indices in Acute Uteroplacental Flow Occlusion

This study was undertaken to investigate in an acute model the effects of acute transient occlusion of the uterine blood flow on the umbilical arterial Doppler indices. Transient uteroplacental circulatory insufficiency was produced in five pregnant ewes (gestational age 115–120 days) by reversible mechanical occlusion of the common hypogastric artery for 20 seconds. The Doppler indices measured were the systolic-diastolic ratio, the pulsatility index and the resistance index. The duration of the cardiac cycle and its systolic and diastolic times were approximated from the Doppler waveform. The occlusion produced transient increases in the Doppler indices and cardiac cycle time. The uteroplacental circulatory insufficiency induced increases (P < 0.005) in the Doppler indices disappeared (P > 0.05) when the latter were corrected for the changes in the cardiac cycle time. Furthermore, diastolic time was the main component of the changes in the cardiac cycle time, and the Doppler indices demonstrated a higher correlation with diastolic time than with systolic time. The results verified that changes in the duration of the fetal cardiac cycle play an important role in shaping the umbilical arterial Doppler waveform and hence need to be taken into account in estimating the Doppler indices.     

MALIGNANT HYPERPYREXIA AND ISOFLURANE: A Case Report

Malignant hyperpyrexia developed, and was successfully treated,in a 50-year old man undergoing pyelolithotomy. Early diagnosiswith the assistance of end-tidal carbon dioxide monitoring facilitatedprompt treatment with i. v. dantrolene. A positive muscle biopsysubsequently confirmed the diagnosis. The only likely triggeringagent used was isoflurane.