RNA folding in Drosophila shows a distance effect for compensatory fitness interactions.

Phylogenetic-comparative analysis was used to construct a secondary structure of Adh precursor messenger RNA (pre-mRNA) in Drosophila. The analysis revealed that the rate of coevolution of base-pairing residues decreases with their physical distance. This result is in qualitative agreement with a model of compensatory fitness interactions which assumes that mutations are individually deleterious but become harmless (neutral) in appropriate combinations. This model predicts that coupled mutations can become fixed in a population under mutation pressure and random genetic drift, when the mutations are closely linked. However, the rate of joint fixation drops as distance between sites increases and recombination breaks up favorable combinations. RNA secondary structure was also used to interpret patterns of linkage disequilibrium at Adh.

Haplotype test reveals departure from neutrality in a segment of the white gene of Drosophila melanogaster.

Restriction map studies previously revealed extensive linkage disequilibria in the transcriptional unit of the white locus in natural Drosophila melanogaster populations. To understand the causes of these disequilibria, we sequenced a 4722-bp region of the white gene from 15 lines of D. melanogaster and 1 line of Drosophila simulans. Statistical tests applied to the entire 4722-bp region do not reject neutrality. In contrast, a test for high-frequency haplotypes ("Haplotype test") revealed an 834-bp segment, encompassing the 3' end of intron 1 to the 3' end of intron 2, in which the structure of variation deviates significantly from the predictions of a neutral equilibrium model. The variants in this 834-bp segment segregate as single haplotype blocks. We proposed that these unusually large haplotype blocks are due to positive selection on polymorphisms within the white gene, including a replacement polymorphism, Arg-->Leu, within this segment.

Multi-locus selection and the structure of variation at the white gene of Drosophila melanogaster.

We surveyed sequence variation and divergence for the entire 5972-bp transcriptional unit of the white gene in 15 lines of Drosophila melanogaster and one line of D. simulans. We found a very high degree of haplotypic structuring for the polymorphisms in the 3' half of the gene, as opposed to the polymorphisms in the 5' half. To determine the evolutionary mechanisms responsible for this pattern we sequenced a 1612-bp segment of the white gene from an additional 33 lines of D. melanogaster from a European and a North American population. This 1612-bp segment encompasses an 834-bp region of the white gene in which the polymorphisms form high frequency haplotypes that cannot be explained by a neutral equilibrium model of molecular evolution. The small number of recombinants in the 834-bp region suggests epistatic selection as the cause of the haplotypic structuring, while an investigation of nucleotide diversity supports a directional selection hypothesis. A multi-locus selection model that combines features from both hypotheses and takes the recent history of D. melanogaster into account may be the best explanation for these data.

Effects of a calcium channel blocker on cardiac output distribution in conscious hypertensive dogs.

The effects of nitrendipine, 8 micrograms/kg/minute, were evaluated in six conscious dogs through measurements of arterial pressure and blood flow in the ascending aorta (cardiac output), mesenteric, renal, and iliac arteries before and after induction of chronic perinephritic hypertension. Before hypertension was induced, nitrendipine reduced mean arterial pressure 19 +/- 2.3% (from 95 +/- 3.2 mm Hg), decreased total peripheral resistance (60 +/- 2.6%), and increased cardiac output (108 +/- 10.5%). These values returned to baseline within 15 to 30 minutes. Nitrendipine caused the greatest increase in blood flow in the iliac bed (98 +/- 9.9%), an intermediate increase in the mesenteric bed (37 +/- 3.7%), and the least increase in the renal bed (7 +/- 2.2%). Two to six weeks after induction of hypertension, administration of nitrendipine elicited significant (p less than 0.01) decreases in mean arterial pressure (32 +/- 2.5% from 151 +/- 4.8 mm Hg) and total peripheral resistance (67 +/- 1.3%) compared with its administration in normotensive dogs, while the increase in cardiac output was not significantly changed (111 +/- 10.9%). These changes in arterial pressure and vascular resistances also were prolonged (i.e., hemodynamics returned to baseline after 75-90 minutes). The increase in iliac (99 +/- 16.8%) and renal (9 +/- 6.1%) blood flows after nitrendipine administration in hypertensive dogs was similar to that found in the normotensive dogs, but mesenteric blood flow doubled (84 +/- 8.4%). Thus, in conscious, hypertensive dogs, nitrendipine administration appears to markedly decrease arterial pressure and total peripheral and regional resistances, which also require more time to return to baseline, but appears to increase blood flow by a greater amount only in the mesenteric bed.

beta-Adrenergic and cholinergic receptors in hypertension-induced hypertrophy.

Perinephritic hypertension was produced in dogs by wrapping one kidney with silk and removing the contralateral kidney 1 week later. Mean arterial pressure rose from 104 +/- 3 to 156 +/- 11 mm Hg, while left ventricular free wall weight, normalized for body weight, was increased by 49%. Muscarinic, cholinergic receptor density measured with [3H]-quinuclidinyl benzilate, fell in hypertensive left ventricles (181 +/- 19 fmol/mg, n = 6; p less than 0.01) as compared with that found in normal left ventricles (272 +/- 16 fmol/mg, n = 8), while receptor affinity was not changed. The beta-adrenergic receptor density, measured by binding studies with [3H]-dihydroalprenolol, rose in the hypertensive left ventricles (108 +/- 10 fmol/mg, n = 7; p less than 0.01) as compared with that found in normal left ventricles (68.6 +/- 5.2 fmol/mg, n = 15), while beta-adrenergic receptor affinity decreased in the hypertensive left ventricles (10.4 +/- 1.2 nM) compared with that found in the normal left ventricles (5.0 +/- 0.7 nM). Plasma norepinephrine levels were similar in the two groups, but myocardial norepinephrine levels were depressed (p less than 0.05) in dogs with hypertension. Moderate left ventricular hypertrophy induced by long-term aortic banding in dogs resulted in elevations in beta-adrenergic receptor density (115 +/- 14 fmol/mg) and decreases in affinity (10.4 +/- 2.2 nM) similar to those observed in the dogs with left ventricular hypertrophy induced by hypertension. Thus, our results suggest that perinephritic hypertension in the dog induces divergent effects on cholinergic and beta-adrenergic receptor density. The increased beta-adrenergic receptor density and decreased affinity may be a characteristic of left ventricular hypertrophy rather than hypertension.

Neuropeptide Y: Y1 and Y2 affinities of the complete series of analogues with single D-residue substitutions.

In an effort to gain insight into the bioactive conformation of neuropeptide Y upon interaction with its receptors, all single-point D-amino acid substituted NPY analogues were prepared, and their Y1 and Y2 receptor binding affinities were evaluated using the human neuroblastoma cell lines, SK-N-MC and SK-N-BE2, respectively. Solid-phase synthesis (Boc strategy) followed by preparative HPLC purification produced analogues of high purity that were characterized by RP-HPLC, AAA, LSIMS, CZE, and optical rotation. Of the 37 isomers (a naturally occurring glycine at position 9 was replaced by Ala and D-Ala), Y1 receptor binding was most perturbed by chiral inversion of residues at the C-terminus (residues 20, 27, 29-35, Ki > or = 300 nM). Substitutions at residues 2-5, 28, and 36 had Ki values ranging from 40 to 260 nM. Substitutions at all other positions yielded analogues with affinities ranging from 1.5 to 20 nM. Binding affinities to the Y2 class of receptors all measured in the low or sub-nanomolar concentrations, with the exception of C-terminally modified isomers (residues 30-35). Only [D-Arg33]- and [D-Gln34]NPY displayed no measurable binding affinity to Y2 receptors at the highest concentration tested (1000 nM). Representative analogues were selected on the basis of their binding affinities and position in the sequence for structural analysis using circular dichroism (CD) spectroscopy. Of the nine peptide evaluated ([D-Pro5]-, [Ala9]-, [D-Glu10]-, [D-Asp11]-, [D-Ala18]-, [D-Tyr20]-, [D-Tyr27]-, and [D-Arg33]NPY), only [D-Tyr27]NPY expressed a definitive correlation between loss of binding affinity and disruption of secondary structure by having the propensity to form beta-sheets at the expense of alpha-helical content. It was concluded that although the incorporation of a single D-amino acid within the sequence of NPY may confer a conformational perturbation, the receptor interaction was only affected when certain critical residues were modified, findings that provide a basis for the identification of the binding pharmacophore of NPY.

Identification of high-potency neuropeptide Y analogues through systematic lactamization.

In the pursuit of potent analogues of neuropeptide Y (NPY) that are selective for the Y1 receptor subtype, two lactam bridge scans of a centrally truncated parent compound were synthesized. A single lactam bridge (gamma-carboxyl of Glu to epsilon-amino of Lys) extending from residues i to i + 3 or i to i + 4 of the proposed alpha-helical region (residues 25-31 of NPY) was introduced in des-AA7-24[Gly6]NPY. Cyclogues (contraction of cyclic analogues), which were approximately one-half the size of native NPY, were initially screened for binding affinity at two discrete NPY receptor types using human neuroblastoma cell membranes, SK-N-MC and SK-N-BE2. Exploitation of the subtle differences present on each receptor type allowed for the identification of cyclogues which bound specifically to Y1 receptors with increased affinity when compared to the corresponding linear parent analogue, while one short Y1 specific cyclogue, des-AA2,3,5,7-24cyclo-(26/29)[Gly6,Glu26,Lys2 9,Pro34]NPY, bound with Ki = 16 nM. Other cyclogues showed distinct preference for Y2 receptors and bound in the low-nanomolar range. Functionally, the compounds inhibited the norepinephrine-stimulated accumulation of cAMP indicating that all acted as agonists with varying potencies.

Design of monocyclic (1-3) and dicyclic (1-3/4-10) gonadotropin releasing hormone (GnRH) antagonists.

Careful analysis of the NMR structures of cyclo(4-10)[Ac-Delta(3)Pro(1),DFpa(2),DTrp(3),Asp(4),DNal (6), Dpr(10)]GnRH, dicyclo(4-10/5-8)[Ac-DNal(1),DCpa(2),DTrp(3), Asp(4), Glu(5),DArg(6),Lys(8),Dpr(10)]GnRH, and dicyclo(4-10/5, 5'-8)[Ac-DNal(1),DCpa(2),DPal(3),Asp(4), Glu(5)(Gly),DArg(6),Dbu(8), Dpr(10)]GnRH showed that, in the N-terminal tripeptide, a type II beta-turn around residues 1 and 2 was probable along with a gamma-turn around DTrp(3)/DPal(3). This suggested the possibility of constraining the N-terminus by the introduction of a cyclo(1-3) scaffold. Optimization of ring size and composition led to the discovery of cyclo(1-3)[Ac-DAsp(1),DCpa(2),DLys(3),DNal(6), DAla(10)]GnRH (5, K(i) = 0.82 nM), cyclo(1,1'-3)[Ac-DAsp(1)(Gly), DCpa(2),DOrn(3),DNal(6),DAla(10)]GnRH (13, K(i) = 0.34 nM), cyclo(1, 1'-3)[Ac-DAsp(1)(Gly),DCpa(2),DLys(3),DNal(6),DA la(10)]GnRH (20, K(i) = 0.14 nM), and cyclo(1,1'-3)[Ac-DAsp(1)(betaAla), DCpa(2), DOrn(3),DNal(6),DAla(10)]GnRH (21, K(i) = 0.17 nM), which inhibited ovulation significantly at doses equal to or lower than 25 microgram/rat. These results were particularly unexpected in view of the critical role(s) originally ascribed to the side chains of residues 1 and 3.(1) Other closely related analogues, such as those where the [DAsp(1)(betaAla), DOrn(3)] cycle of 21 was changed to [DOrn(1)(betaAla), DAsp(3)] of cyclo(1,1'-3)[Ac-DOrn(1)(betaAla), DCpa(2),DAsp(3),DNal(6),DAla(10)]GnRH (22, K(i) = 2.2 nM) or where the size of the cycle was conserved and [DAsp(1)(betaAla), DOrn(3)] was replaced by [DGlu(1)(Gly), DOrn(3)] as in cyclo(1, 1'-3)[Ac-DGlu(1)(Gly),DCpa(2),DOrn(3),DNal(6),DA la(10)]GnRH (23, K(i) = 4.2 nM), were approximately 100 and 25 times less potent in vivo, respectively. Analogues with ring sizes of 18 ¿cyclo(1, 1'-3)[Ac-DGlu(1)(Gly),DCpa(2),DLys(3),DNal(6),DA la(10)]GnRH (24)¿ and 19 ¿cyclo(1,1'-3)[Ac-DGlu(1)(betaAla),DCpa(2),DLys( 3),DNal(6), DAla(10)]GnRH (25)¿ atoms were also less potent than 21 with slightly higher K(i) values (1.5 and 2.2 nM, respectively). These results suggested that the N-terminal tripeptide was likely to assume a folded conformation favoring the close proximity of the side chains of residues 1 and 3. The dicyclic analogue dicyclo(1-3/4-10)[Ac-DAsp(1),DCpa(2),DLys(3),Asp (4),DNal(6), Dpr(10)]GnRH (26) was fully active at 500 microgram, with a K(i) value of 1 nM. The in vivo potency of 26 was at least 10-fold less than that of monocyclic cyclo(1-3)[Ac-DAsp(1),DCpa(2),DLys(3),DNal(6), DAla(10)]GnRH (5); this suggested the existence of unfavorable interactions between the now optimized and constrained (1-3) and (4-10) cyclic moieties that must interact as originally hypothesized. Tricyclo(1-3/4-10/5-8)[Ac-DGlu(1),DCpa(2), DLys(3),Asp(4),Glu(5), DNal(6),Lys(8),Dpr(10)] GnRH (27) was inactive at 500 microgram/rat with a corresponding low affinity (K(i) = 4.6 nM) when compared to those of the most potent analogues (K(i) < 0.5 nM).

Design of potent dicyclic (1-5/4-10) gonadotropin releasing hormone (GnRH) antagonists.

In three earlier papers, the structures and biological potencies of numerous mono- and dicyclic antagonists of GnRH were reported. Among these, two families, each containing two to four members were identified that had very high antagonist potencies in an antiovulatory assay (within a factor of 2 of those of the most potent linear analogues) and high affinities (K(i) < 0.5 nM) for the rat GnRH receptor (rGnRHR). The most favored cycles bridged the side chains of residues (4-10),(1,2) (5-8),(2) (4-10/5-8),(2) (1-3),(3) and (1-3/4-10).(3) Our goal was to identify a consensus model of bioactive conformations of GnRH antagonists, yet these biocompatible constraints did not sufficiently restrain the spatial location of the N-terminal tripeptide with respect to the C-terminal heptapeptide, due largely to the rotational freedom about the bonds connecting these regions. Examination of models derived from NMR studies of cyclo(4-10) analogues suggested a large number of possible cyclic constraints such as cyclo (0-8), (1-8), or (2-8). All analogues tested with these substitutions were inactive as antiovulatory agents at 1 mg/rat (5-9) and had low affinity for rGnRHR. On the other hand, bridging positions 3 and 8 with a [DAsp(3)] to [Dbu(8)] (12, K(i) = 13 nM) or [Orn(8)] (13, K(i) = 14 nM) in the parent compound cyclo(3-8)[Ac-DNal(1),DCpa(2),DXaa(3), Arg(5),DNal(6),Xbb(8),DAla(10)]GnRH yielded analogues that blocked ovulation at 250 microgram/rat. Analogue 14 (K(i) = 2.3 nM), with a [DAsp(3), Lys(8)] bridge, was fully active at 50 microgram/rat. Loss of potency (>20-fold) was observed with the substitution of [DAsp(3)] in 14 by [DGlu(3)] in 15 (K(i) = 23 nM). Dicyclic analogues possessing the (4-10) cycle and selected (1-6), (2-6), and (2-8) cycles led to analogues that were inactive at doses of 500 microgram/rat or larger. Two analogues with (1-8/4-10) cycles (16, K(i) = 1.1 nM) or (3-8/4-10) cycles (22, K(i) = 17 nM) showed full antiovulatory potency at 250 microgram/rat. None of these substitutions yielded analogues potent enough (>80% inhibition of ovulation at 5 microgram/rat or less and K(i) < 0.5 nM) to be candidates for structural analysis by NMR. On the other hand, four dicyclic (1, 1'-5/4-10) analogues met this criterion: dicyclo(1, 1'-5/4-10)[Ac-Asp(1)(Gly),DCpa(2),DTrp(3),Asp(4),Dbu(5 ), DNal(6), Dpr(10)]GnRH (32, K(i) = 0.22 nM), dicyclo(1, 1'-5/4-10)[Ac-Asp(1)(Gly),DCpa(2),DNal(3),Asp(4),Dbu(5 ), DNal(6), Dpr(10)]GnRH (34, K(i) = 0.38 nM), dicyclo(1, 1'-5/4-10)[Ac-Asp(1)(betaAla),DCpa(2), DTrp(3),Asp(4),Dbu(5),DNal(6), Dpr(10)]GnRH (40, K(i) = 0.15 nM), and dicyclo(1, 1'-5/4-10)[Ac-Glu(1)(Gly), DCpa(2),DTrp(3),Asp(4),Dbu(5),DNal(6), Dpr(10)]GnRH (41, K(i) = 0.24 nM). Since they differed slightly in terms of the (1,1'-5) bridge length (21 and 22 atoms) and bridgehead configuration, we may hypothesize that they assume similar bioactive conformations that satisfy a very discriminating receptor, since many other closely related analogues were significantly less potent.

Constrained corticotropin releasing factor antagonists (astressin analogues) with long duration of action in the rat.

In an earlier report we identified specific modifications and substitutions of corticotropin releasing factor (CRF) that led to the discovery of antagonists with extended duration of action as compared to that of astressin {cyclo(30-33)[DPhe(12),Nle(21),Glu(30), Lys(33),Nle(38)]hCRF((12)(-)(41))}. These additional modifications included elongation of the peptide chain by three residues at the N-terminus, its acetylation, and the [CalphaMeLeu(27)] substitution to yield cyclo(30-33)[DPhe(12), Nle(21),CalphaMeLeu(27),Glu(30), Lys(33),Nle(38)]Ac-hCRF((9)(-)(41)), which was found to be longer acting than astressin (Rivier, J.; et al. J. Med. Chem. 1998, 41, 5012-5019). To further increase the efficiency (potency, duration of action, and bioavailability) of this family of antagonists, we introduced two or more CalphaMe-leucine residues at positions shown in earlier studies to be favorable (Hernandez, J.-F.; et al. J. Med. Chem. 1993, 36, 2860-2867). Whereas the introduction of CalphaMe-leucine residues at positions 27 and either 18 (11), 37 (17), or 40 (19) resulted in dramatic increases in duration of inhibitory action in the adrenalectomized (adx) rat after intravenous injection, the same substitution at positions 27 and either 15 (7, 8), 17 (9), 19 (12, 13), or 41 (20) led to short acting analogues. Other substitutions by CalphaMeLeu at positions 27 and either 10 (4), 13 (5), 14 (6), 21 (14), 24 (15), 36 (16), or 38 (18) yielded analogues with duration of action intermediate between those mentioned above. Cyclo(30-33)[DPhe(12), Nle(21), CalphaMeLeu(27),Glu(30),Lys(33),Nle(38), CalphaMeLeu(40)]Ac-hCRF((9)(-)(41)) (astressin B, 19) was one of the most efficacious analogues of this series (>4 h inhibition of ACTH secretion at 25 microgram/adx rat). It was found to be even longer acting via subcutaneous administration in either an aqueous (>24 h inhibition of ACTH secretion at 100 microgram/adx rat) or lipid milieu (DMSO/peanut oil, >24 h inhibition of ACTH secretion at 30 microgram/adx rat) than after intravenous administration (<12 h inhibition of ACTH secretion at 100 microgram/adx rat). We concluded that Calpha-methylation at some positions may favor a bioactive conformation while also preventing degradation and/or elimination, resulting in significant extension of duration of action.

Defining structural requirements for neuropeptide Y receptors using truncated and conformationally restricted analogues.

To further elucidate the minimum bioactive conformation of neuropeptide Y (NPY), a series of truncated and conformationally constrained analogues has been prepared. The synthesis and purification of these peptides was achieved using routine laboratory strategies and techniques. Parent molecules consisted of the native NPY N-terminal 1-4 and C-terminal 25-36 segments, having the residue 5-24 core replaced by either a single flexible omega-aminoalkanoic acid, or a more rigid Pro-Gly or Pro-DAla sequence which was expected to constrain a putative turn, and allow the N- and C-termini to align. Cross-linking between residues 2 and 27 through lactamization using side-chain length and chirality suggested by computer simulations, resulted in cyclo-(2/27)-des-AA7-24[Glu2,Gly6,DDpr27]NPY that exhibited very high affinity (Ki = 0.3 versus 0.3 nM for NPY) for the Y2 receptor using SK-N-BE2 human neuroblastoma cells, yet very low affinity for the Y1 receptor using SK-N-MC human neuroblastoma cells (Ki = 130 versus 2.0 nM for NPY). The added constraint resulting from bridging in this analogue as well as in others suggested that the combination of the deletion of residues 5-24 and the introduction of an internal ring produced exclusive selectivity for the Y2 receptor with little or no loss of affinity. The tolerance of structural recognition was further demonstrated as a second ring was introduced which was expected to constrain the amphiphilic alpha-helix, resulting in the full Y2 agonist dicyclo (2/27,28/32)-des-AA7-24 [Glu2,32,DAla6,DDpr27,Lys28]NPY. Improvement of Y1 binding activity was achieved only by including more residues (des-AA10-17) in the central PP-fold region, while allowing limited flexibility of the termini. Although the length of the bridge seemed to have little effect on binding potency, changes in the location of and chirality at the bridgehead resulted in analogues with different binding affinities. Combination of optimum structural modifications resulted in cyclo-(7/21)-des-AA10-18[Cys7,21]NPY, an analogue shortened by 25% but retaining comparable binding properties to that of native NPY at Y1 and Y2 receptor types (Ki = 5.1 and 1.3 nM, respectively).

Y1 and Y2 receptor selective neuropeptide Y analogues: evidence for a Y1 receptor subclass.

Neuropeptide Y (NPY), a 36-residue polypeptide produced abundantly in both nervous and peripheral tissues, appears to play a significant role in the regulation of diverse biological processes, including feeding behavior and cardiovascular and psychotropic functions. The actions of NPY are mediated through effective binding to specific receptors of which two, designated Y1 and Y2, have been well characterized. A shortened cyclic analogue of NPY, des-AA10-17-cyclo-7/21[Cys7,21]NPY, was shown to retain high affinity for both human neuroblastoma SK-N-MC and SK-N-BE2 cell types (expressing Y1 and Y2 receptors, respectively). Increasing the size of the ring (des-AA10-17-cyclo-2/27[Cys2,27]NPY) in the present study produced a high-affinity analogue (Ki = 3.0 vs 0.3 nM for NPY) that bound exclusively to Y2 receptors. Using the feedback from structure-activity relationships, we also describe the optimization of specific substitutions and bridging arrangements leading to the production of other truncated, high-affinity Y1 selective analogues which bind, as does NPY itself, in the low-nanomolar range. Of greatest significance, des-AA10-17-cyclo-7/21[Cys7,21,Pro34]NPY (11) was found to possess agonistic properties with an affinity comparable to that of the native NPY molecule when tested for its ability to inhibit norepinephrine-stimulated cAMP release in SK-N-MC human neuroblastoma cells. Compound 11 also caused an increase in blood pressure in anesthetized rats. However, in two central nervous system models of Y1 receptor function, stimulation of feeding and anxiolytic activity, this analogue was inactive, which suggests the presence of a new subclass of receptors. In summary, the present results demonstrate that residues 10-17 of NPY are not directly involved in either Y1 or Y2 receptor recognition or activation. This suggests that the selectivity of NPY receptors is highly dependent on subtle conformational changes such as the substitution of residue 34 to a proline or the introduction of intramolecular constraints. Additionally, we have produced an analogue of NPY that selectively activates peripheral NPY Y1 receptors.

Design of potent dicyclic (4-10/5-8) gonadotropin releasing hormone (GnRH) antagonists.

With the ultimate goal of identifying a consensus bioactive conformation of GnRH antagonists, the compatibility of a number of side chain to side chain bridges in bioactive analogues was systematically explored. In an earlier publication, cyclo[Asp(4)-Dpr(10)]GnRH antagonists with high potencies in vitro and in vivo had been identified.(1) Independently from Dutta et al. (2) and based on structural considerations, the cyclic [Glu(5)-Lys(8)] constraint was also found to be tolerated in GnRH antagonists. We describe here a large number of cyclic (4-10) and (5-8) and dicyclic (4-10/5-8) GnRH antagonists optimized for affinity to the rat GnRH receptor and in vivo antiovulatory potency. The most potent monocyclic analogues were cyclo(4-10)[Ac-DNal(1), DFpa(2),DTrp(3),Asp(4),DArg(6),Xaa(10)]GnRH with Xaa = D/LAgl (1, K(i) = 1.3 nM) or Dpr (2, K(i) = 0.36 nM), which completely blocked ovulation in cycling rats after sc administration of 2.5 microgram at noon of proestrus. Much less potent were the closely related analogues with Xaa = Dbu (3, K(i) = 10 nM) or cyclo(4-10)[Ac-DNal(1), DFpa(2),DTrp(3),Glu(4),DArg(6),D/LAgl(10)]GnRH (4, K(i) = 1.3 nM). Cyclo(5-8)[Ac-DNal(1),DCpa(2),DTrp(3),Glu(5),DArg++ +(6),Lys(8), DAla(10)]GnRH (13), although at least 20 times less potent in the AOA than 1 or 2 with similar GnRHR affinity (K(i) = 0.84 nM), was found to be one of the most potent in a series of closely related cyclo(5-8) analogues with different bridge lengths and bridgehead chirality. The very high affinity of cyclo(5,5'-8)[Ac-DNal(1), DCpa(2),DPal(3),Glu(5)(betaAla),DArg(6),(D or L)Agl,(8)DAla(10)]GnRH 14 (K(i) = 0.15 nM) correlates well with its high potency in vivo (full inhibition of ovulation at 25 microgram/rat). Dicyclo(4-10/5-8)[Ac-DNal(1),DCpa(2),DTrp(3),Asp (4),Glu(5),DArg(6), Lys(8),Dpr(10)]GnRH (24, K(i) = 0.32 nM) is one-fourth as potent as 1 or 2, in the AOA; this suggests that the introduction of the (4-10) bridge in 13, while having little effect on affinity, restores functional/conformational features favorable for stability and distribution. To further increase potency of dicyclic antagonists, the size and composition of the (5-8) bridge was varied. For example, the substitution of Xbb(5') by Gly (30, K(i) = 0.16 nM), Sar (31, K(i) = 0.20 nM), Phe (32, K(i) = 0.23 nM), DPhe (33, K(i) = 120 nM), Arg (36, K(i) = 0.20 nM), Nal (37, K(i) = 4.2 nM), His (38, K(i) = 0.10 nM), and Cpa (39, K(i) = 0.23 nM) in cyclo(4-10/5,5'-8)[Ac-DNal(1),DCpa(2),DPal(3),Asp(4),G lu(5)(Xbb(5')), DArg(6),Dbu,(8)Dpr(10)]GnRH yielded several very high affinity analogues that were 10, ca. 10, 4, >200, 1, ca. 4, >2, and 2 times less potent than 1 or 2, respectively. Other scaffolds constrained by disulfide (7, K(i) = 2.4 nM; and 8, K(i) = 450 nM), cyclo[Glu(5)-Aph(8)] (16, K(i) = 20 nM; and 17, K(i) = 0.28 nM), or cyclo[Asp(5)-/Glu(5)-/Asp(5)(Gly(5'))-Amp(8)] (19, K(i) = 1.3 nM; 22, K(i) = 3.3 nM; and 23, K(i) = 3.6 nM) bridges yielded analogues that were less potent in vivo and had a wide range of affinities. The effects on biological activity of substituting DCpa or DFpa at position 2, DPal or DTrp at position 3, and DArg, DNal, or DCit at position 6 are also discussed. Interestingly, monocyclo(5-8)[Glu(5), DNal(6),Lys(8)]GnRH (18, K(i) = 1. (ABSTRACT TRUNCATED)

Changes in ventricular fibrillation threshold during the development of perinephritic hypertension.

Established hypertension is associated with enhanced susceptibility to life-threatening arrhythmias. However, little is known about the effects of the development of hypertension on the electro-physiological properties of the heart. To study this relationship, dogs were chronically instrumented for recording mean arterial pressure and conducting cardiac electrical testing during right ventricular pacing using an intracavitary bipolar catheter. In normotension, ventricular fibrillation threshold was determined under alpha-chloralose anesthesia, after which perinephritic hypertension was induced by wrapping one kidney in silk followed by contralateral nephrectomy. During serial testing of the same animals, ventricular fibrillation threshold was significantly increased early in the development of hypertension (2 to 3 weeks after renal wrapping), but found to be significantly reduced at 6 to 7 weeks after renal wrapping. The increase in ventricular fibrillation threshold was eliminated by cholinergic blockade.

Plasma norepinephrine changes after arousal and exercise were enhanced in conscious pigs with perinephritic hypertension.

This study found that pigs in the early stages of perinephritic hypertension had enhanced plasma norepinephrine (NE) and heart rate (HR) responses to behaviorally arousing stimuli (lifting in a canvas sling) and exercise. In the hypertensive animals, the increase in plasma NE in response to behavioral arousal caused by lifting was double that of the intact group, and the HR response was 50% greater. The increase in plasma NE in response to exercise was six times greater in hypertensive pigs. By contrast, mean arterial pressure changes were not enhanced in the hypertensive group. Exaggerated catecholamine responses in plasma to lifting and exercise stimulation were present when hemodynamic responses were not exaggerated.

Intracerebroventricular propranolol prevented vascular resistance increases on arousal from sleep apnea.

Despite the increased risk of sudden cardiac death associated with sleep apnea, little is known about mechanisms controlling cardiovascular responses to sleep apnea and arousal. Chronically instrumented pigs were used to investigate the effects of airway obstruction (AO) during rapid-eye-movement (REM) and non-REM (NREM) sleep and arousal on mean arterial pressure (MAP), heart rate (HR), cardiac output (CO), and total peripheral resistance (TPR). A stainless steel cannula was implanted in the lateral cerebral ventricle. During REM sleep, HR was 133 +/- 10 beats/min, MAP was 65 +/- 8 mmHg, CO was 1,435 +/- 69 ml/min, and TPR was 0.046 +/- 0.004 mmHg.ml-1.min. During AO, CO decreased by 90 +/- 17 ml/min (P < 0.05). On arousal from AO, MAP increased by 15 +/- 3 mmHg, HR increased by 10 +/- 3 beats/min, and TPR increased by 0.008 +/- 0.001 mmHg.ml-1.min (all P < 0.05). Changes during NREM were similar but were more modest during AO. After the intracerebroventricular administration of propranolol (50 micrograms/kg; a beta-adrenoreceptor blocking agent), decreases in CO during AO and increases in HR during arousal were intact, but increases in MAP and TPR were no longer significant. These data suggest that vascular responses to AO during sleep may be regulated in part by beta-adrenergic receptors in the central nervous system.

Hemodynamic changes associated with obstructive sleep apnea followed by arousal in a porcine model.

To study the effects of airway obstruction (AWO) and arousal on coronary blood flow, mean arterial pressure (MAP), and heart rate, pigs were chronically instrumented with arterial catheters, Doppler flow probes on the left circumflex coronary artery, and electrodes for determination of sleep stages. A modified tracheostomy tube was placed in the trachea to obstruct the upper airway during sleep sessions. In control studies, during non-rapid-eye-movement (NREM) sleep, MAP was 84 +/- 2 mmHg before AWO and increased by 5 +/- 2 mmHg on arousal. MAP was lower during rapid-eye-movement (REM) sleep (62 +/- 2 mmHg), and the increase on arousal was fourfold greater (22 +/- 2 mmHg). Heart rate was similar in both sleep stages (NREM: 120 +/- 4 beats/min; REM: 124 +/- 5 beats/min) and increased significantly on arousal (NREM: 12 +/- 2 beats/min; REM: 18 +/- 1 beats/min). Coronary blood flow was similar during both stages (NREM: 43 +/- 4 ml/min; REM: 46 +/- 8 ml/min) and increased by 12-15% on arousal. Coronary vascular resistance index increased significantly by 24% on arousal from AWO during REM sleep. All increases and decreases were significant at P < 0.05. Receptor blockade studies were performed to assess alpha-adrenergic receptor involvement.

Cardiovascular responses to nonrespiratory and respiratory arousals in a porcine model.

Spontaneous and provoked nonrespiratory arousals can be accompanied by a patterned hemodynamic response. To investigate whether a patterned response is also elicited by respiratory arousals, we compared nonrespiratory arousals (NRA) to respiratory arousals (RA) induced by airway occlusion during non-rapid eye movement sleep. We monitored mean arterial blood pressure (MAP), heart rate, iliac and renal blood flow, and sleep stage in 7 pigs during natural sleep. Iliac and renal vascular resistance were calculated. Airway occlusions were obtained by manually inflating a chronically implanted tracheal balloon during sleep. The balloon was quickly deflated as soon as electroencephalogram arousal occurred. As previously reported, NRA generally elicited iliac vasodilation, renal vasoconstriction, little change in MAP, and tachycardia. In contrast, RA generally elicited iliac and renal vasoconstriction, an increase in MAP and tachycardia. The frequent occurrence of iliac vasoconstriction and arterial pressure elevation following RA but not NRA suggests that sleep state change alone does not account for the hemodynamic response to airway occlusion during sleep.

Patterned cardiovascular responses to sleep and nonrespiratory arousals in a porcine model.

Patients with obstructive sleep apnea experience marked cardiovascular changes with apnea termination. Based on this observation, we hypothesized that sudden sleep disruption is accompanied by a specific, patterned hemodynamic response, similar to the cardiovascular defense reaction. To test this hypothesis, we recorded mean arterial blood pressure, heart rate, iliac blood flow and vascular resistance, and renal blood flow and vascular resistance in five pigs instrumented with chronic sleep electrodes. Cardiovascular parameters were recorded during quiet wakefulness, during non-rapid-eye-movement and rapid-eye-movement sleep, and during spontaneous and induced arousals. Iliac vasodilation (iliac vascular resistance decreased by -29.6 +/- 4.1% of baseline) associated with renal vasoconstriction (renal vascular resistance increased by 10.3 +/- 4.0%), tachycardia (heart rate increase: +23.8 +/- 3.1%), and minimal changes in mean arterial blood pressure were the most common pattern of arousal response, but other hemodynamic patterns were observed. Similar findings were obtained in rapid-eye-movement sleep and for acoustic and tactile arousals. In conclusion, spontaneous and induced arousals from sleep may be associated with simultaneous visceral vasoconstriction and hindlimb vasodilation, but the response is variable.

Analogs of thyrotropin-releasing hormone using an aminoglycine-based template.

Novel thyrotropin-releasing hormone (TRH, pGlu-His-Pro-NH2) analogs, made by solid phase, were derived from the general scaffold pGlu-(D/L)Agl(X)-Pro-NH2 where Agl = aminoglycine. Analogs ranged from X being a proton to an acylating agent derived from substituted (aromatic heterocyclic rings) formic or acetic acids or an aminotriazolyl moiety (3'-amino-1H-1',2',4'-triazolyl) built on N(alpha) of aminoglycine or Nbeta of alpha,beta-diaminoproprionic acid (Dpr). X was expected to mimic the electronic and structural characteristics of the imidazole ring of histidine. Analogs were purified by HPLC, characterized by mass spectrometry and isolated as either diastereoisomeric mixtures or pure isomers. Analogs, tested for their binding affinity to mouse pituitary TRH receptors, have apparent equilibrium inhibitory constants > 1 microM.