Parallel production of oligonucleotide arrays using membranes and reagent jet printing.

Here, we present a low-cost method to produce compact arrays using microporous materials and reagent jetting. Oligonucleotides are immobilized on membrane sheets as a series of lines. The membrane sheet is then rolled and bound, and the spiral structure is cut like a "jelly roll" to produce identical arrays. The spiral arrays behave much like larger formats using membranes, and hybridization detection can be accomplished using standard signal-generation mechanisms. The method is particularly useful for producing identical arrays from pre-synthesized oligonucleotides.

Synthesis and in vitro characterization of N-[5-(4,5-dihydro-1H-imidazol-2-yl)-2-hydroxy-5,6,7,8- tetrahydronaphthalen-1-yl]methanesulfonamide and its enantiomers: a novel selective alpha 1A receptor agonist.

The existence of multiple subtypes of the alpha 1 adrenergic receptor has been demonstrated both pharmacologically and by molecular biological cloning techniques. The development of subtype selective antagonists has been the focus of much research within the pharmaceutical industry, and clinical evidence now exists that alpha-1A selective antagonists will have utility in the treatment of benign prostatic hyperplasia. However, highly subtype selective agonists are not known. Herein we report the synthesis and pharmacological characterization of N-[5-(4,5-dihydro-1H-imidazol-2-yl)-2-hydroxy-5,6,7,8- tetrahydronaphthalen-1-yl]methanesulfonamide and its enantiomers, a highly potent full agonist with excellent selectivity for the alpha 1A receptor subtype.

Alpha 1-adrenoceptor-induced contractility in rat aorta is mediated by the alpha 1D subtype.

Adrenoceptor agonists were used to characterize the alpha 1-adrenoceptor subtype responsible for mediating tension (phasic and tonic combined) in the denuded rat aorta and compared with radioligand binding at alpha 1-adrenoceptor subtypes. The rank order of potency at the rat aorta was the same as that obtained for binding affinity at the rat clonal alpha 1d-adrenoceptor: norepinephrine > epinephrine > cirazoline > phenylephrine > oxymetazoline > A-61603 > methoxamine. Correlation coefficients comparing rat aortic contraction (pD2) to binding (pKi) were 0.09-0.21 for alpha 1A/a receptors, 0.66 for clonal alpha 1b and 0.94 for clonal alpha 1d-adrenoceptors. Correlation coefficients comparing the clonal alpha 1d-adrenoceptor binding affinity to in vitro contractile responses were 0.03 and 0.10 for the rat vas deferens and canine prostate alpha 1A-adrenoceptor responses, respectively, 0.09 for the rat spleen alpha 1B and as noted, 0.94 for the rat aorta. The agreement observed between agonist potency at the rat aorta and affinity for the alpha 1d binding site provide new evidence that the alpha 1D-adrenoceptor subtype is responsible for mediating contractions in the rat aorta.

Actions of terazosin and its enantiomers at subtypes of alpha 1- and alpha 2-adrenoceptors in vitro.

Terazosin and its enantiomers, antagonists of alpha 1-adrenoceptors, were studied in radioligand binding and functional assays to determine relative potencies at subtypes of alpha 1- and alpha 2-adrenoceptors in vitro. The racemic compound and its enantiomers showed high and apparently equal affinity for subtypes of alpha 1-adrenoceptors with Kl values in the low nanomolar range, and showed potent antagonism of alpha 1-adrenoceptors in isolated tissues, with the enantiomers approximately equipotent to the racemate at each alpha 1-adrenoceptor subtype. At alpha 2b sites, R(+) terazosin bound less potently than either the S(-) enantiomer or racemate. R(+) terazosin was also less potent than the S(-) enantiomer or the racemate at rat atrial alpha 2B receptors. These agents were not significantly different in their potencies at alpha 2a or alpha 2A sites. Since the high affinity for alpha 2B sites of quinazoline-type alpha-adrenoceptor antagonists has been used to differentiate alpha 2-adrenoceptor subtypes, the low affinity of R(+) terazosin for these sites was unexpected. Because terazosin or its enantiomers are approximately equipotent at alpha 1-adrenoceptor subtypes, the lower potency of R(+) terazosin at alpha 2B receptors indicates a somewhat greater selectivity for alpha 1-compared to alpha 2B adrenoceptor subtypes. The possible pharmacological significance of this observation is discussed.

A-61603, a potent alpha 1-adrenergic receptor agonist, selective for the alpha 1A receptor subtype.

N-[5-(4,5-dihydro-1H-imidazol-2yl)-2-hydroxy-5,6,7,8-tetrahydro naphthalen-1-yl] methanesulfonamide hydrobromide (A-61603) is a novel and potent alpha-adrenoceptor agonist. In radioligand binding assays, the compound is at least 35-fold more potent at alpha 1A/a receptors than at alpha 1b or alpha 1d sites. In fibroblast cells transfected with alpha 1a receptors, A-61603 more potently stimulates phosphoinositide hydrolysis than norepinephrine, and is antagonized by prazosin. A-61603 is less potent in cells transfected with alpha 1b or alpha 1d receptors. A-61603 is a potent agonist at alpha 1A receptors in rat vas deferens (200- to 300-fold more potent than norepinephrine or phenylephrine, respectively) and in isolated canine prostate strips (130- to 165-fold more potent than norepinephrine or phenylephrine, respectively). In contrast, A-61603 is only 40-fold more potent than phenylephrine at alpha 1B sites in rat spleen and 35-fold less potent at rat aortic, alpha 1D sites. In an in vivo dog model, A-61603 raises intraurethral prostatic tone to a greater extent than mean arterial blood pressure. A-61603 induces a pressor response in conscious rats at doses 50- to 100-fold lower than phenylephrine, and the response is not attenuated by pretreatment with CEC, whereas YM-617 causes a 100-fold shift in the response. These results indicate that A-61603 is a potent adrenergic agonist, selective for alpha 1A/a receptors, and may prove a useful probe for studies of adrenergic function and alpha 1 adrenoceptor regulation of physiological functions.

Inhibition of norepinephrine transport into synaptic vesicles by amphetamine analogs.

The abilities of several amphetamine analogs with restricted conformations to inhibit uptake of [3H]norepinephrine into synaptic vesicles isolated from rat brain cerebral cortex were compared. [3H]Norepinephrine was accumulated in the vesicles with a Km of 3.5 microM and a Vmax of 7.6 pmol/g of tissue per min. This uptake was inhibited by reserpine (IC50, 6.4 nM), amphetamine (IC50, 2.5 microM) and eight amphetamine analogs. 2-Aminotetralin, the most flexible of the analogs (capable of assuming both gauche and anticonformations), was the most potent (IC50, 22 microM). The side chain of amphetamine was held in one of its two low energy conformations [transantiperiplanar (extended) and gauche (folded)]. This was accomplished by using the benzobicyclo[2.2.1]heptane, benzobicylco[2.2.2]octane, or tetrahydroisoquinoline ring systems. The potencies of all of the conformationally defined analogs were reduced with IC50 values of 120 to 370 microM and the potency differences between anti- and gauche conformations were small. These results are in contrast to those obtained by us earlier for inhibition of neuronal reuptake and suggest that vesicular uptake may be more conformationally restrictive than neuronal reuptake. It is possible that: 1) the amphetamine pharmacophore must retain some conformational flexibility for vesicular uptake (hence activity for 2-aminotetralin but not for the rigid analogs); 2) there is another higher energy conformation of amphetamine not present in any of the rigid analogs evaluated that is required for optimal interaction with the vesicular uptake site; or 3) the extra steric bulk of the bridging atoms in the conformational analogs severely interferes with binding at the vesicular uptake site.

Effects of calcium depletion on norepinephrine- and A23187-induced stimulation of inositol phosphate formation.

The role of calcium in the stimulation of phosphoinositide (PIn) hydrolysis by norepinephrine and the calcium ionophore A23187 was investigated in chopped cerebral cortex in which the PIns had been labeled previously with ([3H]myo-inositol. The accumulation of the newly formed [3H]inositol phosphate ([3H]IPs) was used as an index of PIn hydrolysis. Norepinephrine produced a concentration-related increase in the accumulation of [3H]inositol-phosphates ([3H]IP), and this effect was only partially antagonized by omission of calcium from the incubation medium. Norepinephrine had relatively little effect on the accumulation of [3H]inositol 1,4-bisphosphate [3H]IP2 and inositol 1,4,5-triphosphate and/or inositol 1,3,4-trisphosphate ([3H]IP3). A23187 also increased the accumulation of [3H]IP but this effect was not antagonized by omission of calcium from the incubation medium. When the tissue had been washed extensively with EGTA, the basal levels of [3H]IP, [3H]IP2 and [3H]IP3 were decreased markedly, and the effects of both norepinephrine and A23187 were antagonized. Addition of calcium back to the depleted tissue led to an increase in the basal level of [3H]IPs as well as restoration of the stimulation produced by norepinephrine and A23187. The calcium threshold for the PIn effect was 0.1 microM. Additional calcium did not affect markedly the stimulation of accumulation of [3H]IP produced by norepinephrine and A23187. The results suggest that there is an absolute calcium requirement for PIn phosphodiesterase which is attained at 0.1 microM Ca2+. A23187 can stimulate the accumulation of [3H]IP perhaps by providing access of another form of the enzyme to artificially high concentrations (millimolar range) of calcium.

Norepinephrine stimulation of phosphoinositide hydrolysis in rat cerebral cortex is associated with the alpha1-adrenoceptor.

Norepinephrine (NE) and the selective alpha1-adrenoceptor agonist phenylephrine (PE) both markedly stimulate the formation of [3H]inositol phosphates in a concentration-dependent manner upon incubation with [3H]myo-inositol. The selective alpha2 agonist, clonidine, did not significantly alter [3H]inositol phosphate formation, even at concentrations as high as 10(-3) M. The alpha1 antagonist prazosin (IC50, 0.036 microM) was 300 times more potent than the alpha2 antagonist yohimbine (IC50, 10.7 microM) as an inhibitor of NE (10(-4) M)-stimulated phosphatidylinositol (PI) hydrolysis. These results indicate that the alpha1-, but not the alpha2-adrenoceptor subtype in rat brain is coupled to phosphoinositide hydrolysis.