Michael W. Wolowyk

Volume-Sensitive Taurine Transport in Fish Erythrocytes

SummaryTaurine plays an important role in cell volume regulation in both vertebrates and invertebrates. Erythrocytes from two euryhaline fish species, the eel (Anguilla japonica) and the starry flounder (Platichthys stellatus) were found to contain high intracellular concentrations of this amino acid (≃ 30 mmol per liter of cell water). Kinetic studies established that the cells possessed a saturable high-affinity Na+-dependent β-amino-acid transport system which also required Cl− for activity (apparentKm (taurine) 75 and 80 μm;Vmax 0.85 and 0.29 μmol/g Hb per hr for eel (20°C) and flounder cells (10°C), respectively. This β-system operated with an apparent Na+/Cl−/taurine coupling ratio of 2∶1∶1. A reduction in extracellular osmolarity, leading to an increase in cell volume, reversibly decreased the activity of the transporter. In contrast, low medium osmolarity stimulated the activity of a Na+-independent nonsaturable transport route selective for taurine, γ-amino-n-butyric acid and small neutral amino acids, producing a net efflux of taurine from the cells. Neither component of taurine transport was detected in human erythrocytes. It is suggested that these functionally distinct transport routes participate in the osmotic regulation of intracellular taurine levels and hence contribute to the homeostatic regulation of cell volume. Volume-induced increases in Na+-independent taurine transport activity were suppressed by noradrenaline and 8-bromoadenosine-3′, 5′-cyclic monophosphate, but unaffected by the anticalmodulin drug, pimozide.

Synthesis and Calcium Channel Antagonist Activity of 3-Arylmethyl 5-Isopropyl l,4-Dihydro-2,6-dimethyl-4-(pyridyl)-3,5-pyridinedicarboxylates

Unsymmetrical aryl(heteroaryl)methyl isopropyl ester analogues of nifedipine, in which the 2-nitrophenyl group at C-4 is replaced by a 2- or 3-pyridyl substituent, were synthesized and evaluated as calcium-channel antagonists using guinea pig ileal longitudinal smooth muscle. The point of attachment of the C-4 pyridyl substituent was a determinant of activity where the relative potency order was 2-pyridyl > 3-pyridyl. Within the C-4 2-pyridyl series of compounds, an electronegative substituent such as a trifluoromethyl or bromo at the 4 position of the benzyl ester substituent or a nitrogen atom at the 1 position of a 4-pyridylmethyl ester substituent, enhanced activity relative to the unsubstituted benzyl ester analogue. In contrast, in the C-4 3-pyridyl class of compounds, a variety of aryl(heteroaryl)methyl ester substituents did not alter potency to any significant extent. A number of compounds in the C-4 2-pyridyl series possessing 4-pyridylmethyl, 4-trifluoromethylbenzyl, 4-bromobenzyl, and 3-pyridylmethyl ester substituents were approximately equipotent to nifedipine. The aryl(heteroaryl)methyl ester and C-4 2-pyridyl substituents therefore appear to provide important interdependent contributions to calcium-channel antagonist activity.

Nucleoside uptake by red blood cells from a primitive vertebrate, the pacific hagfish (Eptatretus stouti), is mediated by a nitrobenzylthioinosine-insensitive transport system

Red blood cells from the Pacific hagfish (Eptatretus stouti) were found to possess a facilitated diffusion nucleoside transport system insensitive to inhibition by the nucleoside transport inhibitor nitrobenzylthioinosine (NBMPR). Uridine uptake by this route was saturable (apparent Km 0.14 mM; Vmax 2 mmol/l cells per h at 10 degrees C), inhibited by inosine and adenosine, and blocked both by the vasodilator dipyridamole and by the thiol-reactive agent p-chloromercuriphenylsulphonate. The properties of this carrier resemble closely those of NBMPR-insensitive nucleoside transport systems in some mammalian neoplastic cell lines and in rat red cells. The presence of this type of carrier in a primitive vertebrate suggests that such transporters have a broad biological distribution and that they pre-date or arose at an early stage of vertebrate evolution.

Synthesis and calcium channel antagonist activity of dialkyl hexahydro-1,2′,6′-trimethyl[bipyridine]-3′,5′-dicarboxylates

Abstract Reaction of dialkyl 1′,4′-dihydro-2′,6′-dimethyl[bipyridine]-3′,5′-dicarboxylates 2 with methyl iodide yielded the corresponding 4′-(1-methylpyridinium) iodide salts 3 in quantitative yield. The sodium borohydride reduction of 3 in aqueous ethanol gave the corresponding dialkyl hexahydro-1,2′,6′-trimethyl[bipyridine]-3′,5′-dicarboxylate analogs 4–5. The calcium channel antagonist activities for 4–5 were determined using the muscarinic receptor-mediated Ca2+-dependent contraction of guinea pig ileal longitudinal smooth muscle. The relative activities for the 3′,5′-diethyl series 5 was 3-tetrahydropyridinyl 5b > 4-tetrahydropyridinyl 5c > 2-tetrahydropyridinyl 5a. Increasing the size of the 3′,5′-alkyl substituents enhanced activity. An approximate 1:1 correlation between the IC50 value for the inhibition of [3H]nitrendipine binding and inhibition of the tonic component of the muscarinic-induced contractile response was observed for 4a and 5b. NMR studies suggest that the 4′-[2-(1-methyl-1,2,3,6-tetrahydropyridinyl)] ring systems of 4a and 5a are anti-periplanar to the 1,4-dihydropyridine ring system.

Synthesis and calcium channel antagonist activity of dialkyl 1,4-dihydro-2,6-dimethyl-4-[4-(1-methoxycarbonyl-1,4-dihydropyridyl)]-3 ,5- pyridinedicarboxylates.

A novel class of dialkyl 1,4‐dihydro‐2,6‐dimethyl‐4‐[4‐(1‐methoxycarbonyl‐ 1,4‐dihydropyridyl)]‐3,5‐pyridinedicarboxylates (8—14) were synthesized and evaluated as calcium channel antagonists. The differences in activity among members of this new class of compounds was less than one log unit (IC50 range of 1.12 × 10‐6 to 8.57 × 10‐6 M), relative to the reference drug nifedipine (IC50 = 1.43 × 10‐8 M). The small differences in potency, irrespective of the size of the dialkyl (Me, Et, i‐Pr, i‐Bu) ester substituents, is attributed to the fact that the N‐CO2Me substituent is too far removed from the C‐3 and C‐5 ester substituents to undergo non‐bonded steric interactions. The 4‐[4‐(1‐methoxycarbonyl‐1,4‐dihydropyridyl) moiety in this new class of compounds is bioisosteric with a C‐4 4‐nitrophenyl, or a 4‐pyridyl, substituent in classical 1,4‐dihydropyridines.