Peripheral changes above and below injury level lead to prolonged vascular responses following high spinal cord injury.

Autonomic dysreflexia (AD) is a debilitating disorder producing episodes of extreme hypertension in patients with high-level spinal cord injury (SCI). Factors leading to AD include loss of vasomotor baroreflex control to regions below injury level, changes in spinal circuitry, and peripheral changes. The present study tested for peripheral changes below and above injury level 6 wk after a transection at the fourth thoracic spinal level. Changes in vascular conductance were recorded in the femoral, renal, brachial, and carotid arteries in response to intravenous injections of two alpha-adrenergic agonists, phenylephrine (PE; 0.03-100 microg/kg) and methoxamine (Meth; 1-300 microg/kg). Unlike PE, Meth is not subject to neuronal reuptake. Ganglionic blockade (0.6 mg/kg chlorisondamine) was used to eliminate the central component of the cardiovascular response. After ganglionic blockade, SCI animals exhibited prolonged vasoconstriction in response to PE in all blood vessels measured compared with those in intact animals (all, P < 0.035). However, the PE dose-response curves obtained after ganglionic blockade revealed no significant difference in the potency between the two groups (all, P > 0.06), indicating that the prolonged vasoconstriction was not due to supersensitivity to PE. In contrast to PE, vascular responses to Meth did not vary between intact and SCI groups (all P > 0.108). These results show the development of a widespread peripheral change producing prolonged vasoconstriction in response to PE, but not Meth, possibly due to reduced neuronal reuptake of PE after SCI. This is the first study to report such a change in blood vessels not only below but also above injury level. Interventions to correct this reduced reuptake may help limit the development of AD.

Sodium transport across the chorioallantoic membrane of porcine placenta involves the epithelial sodium channel (ENaC).

The properties of chorioallantoic membrane derived from Large White Landrace sows at 45, 65 and 100 days gestation are examined. Under short circuit conditions positive charge flows from fetal to maternal sides of the tissue. Na+ is shown to be the sole charge carrier as the short circuit current is inhibited reversibly by fetal applications of amiloride and replacement of Na+ by choline in the Ringer solution, and irreversibly by both fetal and maternal applications of ouabain. The initial short circuit current is smaller at day 100 compared to days 45 and 65. The dose responses to amiloride indicate that the epithelial sodium channel (ENaC) is involved in the movement of Na+ and that it is accessible on the fetal side of the tissue only. Immunostaining shows that the ENaC-alpha subunit is present in both the allantoic membrane and the trophoblast. Uptake studies using microvillous (apical) membrane vesicles suggest it is either inactive or only weakly active at this site. The trophoblast at day 100 has a higher content of ENaC than at days 45 and 65. This is the first report of the presence of ENaC in placental tissues. The effects of ouabain indicate the presence of a Na+ pump that is more readily inhibited by applications of the drug on the maternal aspect of the tissue than on the fetal side. Differential mechanisms may be present that would allow net movement of Na+ in either direction across the chorioallantoic membrane according to the changing demands of the developing fetus.

Patterns of fetal growth within Large White x Landrace and Chinese Meishan gilt litters at three stages of gestation.

Low birthweight piglets have an increased incidence of mortality and morbidity. As there are few opportunities to remedy the detrimental consequences of low birthweight after birth, it is important to understand the nature of fetal growth retardation and to identify when low birthweight fetuses deviate from the growth trajectory of their normally grown siblings. The aims of this study were to identify the nature, timing and possible causal factors influencing inadequate fetal growth in Large White x Landrace (LW) and Chinese Meishan (MS) gilts at three stages of pregnancy. Thirty-six per cent of litters contained inadequately grown fetuses. Both intrauterine-growth-restricted (IUGR) and small-for-gestational-age (SGA) fetuses could be identified as early as Day 30 in MS and LW litters and the percentage of litters containing inadequately grown fetuses was similar throughout gestation. MS fetuses, placentas and piglets had less within-litter variation in weight at all stages studied. Inverse relationships were observed between litter size and both minimum and mean weights of MS neonates. No other relationships between fetal size and either uterine position or litter size were observed.

Causes and consequences of fetal growth retardation in pigs.

In pigs, as in other species, fetal growth retardation is associated with reduced birth weight and increased risk of fetal and neonatal death. As there are few opportunities after birth to remedy the detrimental effects of low birth weight, it is important to understand both the intrinsic and extrinsic factors associated with inadequate fetal growth and to determine when growth retarded fetuses deviate from the growth trajectory of their normal sized littermates. Inadequately grown pig fetuses can be identified statistically as early as day 30 of the 114 days of gestation, indicating that limited uterine space is not a primary determinant of fetal growth. Comparisons of the smallest fetus within a litter with a normal sized sibling reveal that inadequately grown fetuses have altered endocrine status and lower circulating concentrations of many essential amino acids. In addition, the placenta supplying the smallest fetus is disproportionately small and has a reduced capacity to transport amino acids. Understanding the timing and the causes of fetal growth retardation in pigs may help us to devise appropriate strategies to reduce the incidence and hence the detrimental postnatal consequences of runting.

Pharmacological characterization of antagonists of the C5a receptor.

1. Potent and highly selective small molecule antagonists have recently been developed by us for C5a receptors (C5aR) on human polymorphonuclear leukocytes (PMN). In this study we compared a new cyclic antagonist, F-[OPdChaWR], with an acyclic derivative, MeFKPdChaWr, for their capacities to bind to C5aR on human PMN and human umbilical artery membranes. We also compared their inhibition of myeloperoxidase (MPO) secretion from human PMNs and their inhibition of human umbilical artery contraction induced by human recombinant C5a. 2. In both PMNs and umbilical artery, the cyclic and acyclic C5a antagonists displayed insurmountable antagonism against C5a. There were differences in selectivities for the C5aR with F-[OPdChaWR] (pKb 8.64+/-0.21) being 30 times more potent than MeFKPdChaWr (pKb 7.16+/-0.11, P<0.05) in PMNs, but of similar potency (pKb 8.19+/-0.38 vs pKb 8.28+/-0.29, respectively) in umbilical artery. This trend was also reflected in their relative binding affinities, both antagonists having similar affinities (-logIC50 values) for C5aR in umbilical artery membranes (F-[OPdChaWR], 7.00+/-0.46; MeFKPdChaWr, 7.23+/-0.17), whereas in PMN membranes the C5aR affinity of the cycle F-[OPdChaWR] (7.05+/-0. 06) was four times higher than that of acyclic MeFKPdChaWr (6.43+/-0. 24, P<0.05). 3. In summary, the results reveal that these antagonists are insurmountable in nature against C5a for C5aR on at least two human cell types, and the differences in relative receptor binding affinities and antagonistic potencies against C5a are consistent with differences in receptors within these cell types. The nature of these differences is yet to be elucidated.

Low-molecular-weight peptidic and cyclic antagonists of the receptor for the complement factor C5a.

Activation of the human complement system of plasma proteins during immunological host defense can result in overproduction of potent proinflammatory peptides such as the anaphylatoxin C5a. Excessive levels of C5a are associated with numerous immunoinflammatory diseases, but there is as yet no clinically available antagonist to regulate the effects of C5a. We now describe a series of small molecules derived from the C-terminus of C5a, some of which are the most potent low-molecular-weight C5a receptor antagonists reported to date for the human polymorphonuclear leukocyte (PMN) C5a receptor. 1H NMR spectroscopy was used to determine solution structures for two cyclic antagonists and to indicate that antagonism is related to a turn conformation, which can be stabilized in cyclic molecules that are preorganized for receptor binding. While several cyclic derivatives were of similar antagonistic potency, the most potent antagonist was a hexapeptide-derived macrocycle AcF[OPdChaWR] with an IC50 = 20 nM against a maximal concentration of C5a (100 nM) on intact human PMNs. Such potent C5a antagonists may be useful probes to investigate the role of C5a in host defenses and to develop therapeutic agents for the treatment of many currently intractable inflammatory conditions.

Effects of a new C5a receptor antagonist on C5a- and endotoxin-induced neutropenia in the rat.

A new C5a receptor antagonist, the cyclic peptide Phe-[Orn-Pro-D-cyclohexylalanine-Trp-Arg], (F-[OPdChaWR]), was tested for its ability to antagonize the neutropenic effects of both C5a and endotoxin in rats. Human recombinant C5a (2 microg kg(-1) i.v.) caused rapid neutropenia, characterized by an 83% decrease in circulating polymorphonuclear leukocytes (PMNs) at 5 min. Administration of F-[OPdChaWR] (0.3-3 mg kg(-1) i.v.), did not affect the levels of circulating PMNs but, when given 10 min prior to C5a, it inhibited the C5a-induced neutropenia by up to 70%. Administration of E. Coli lipopolysaccharide (LPS, 1 mg kg(-1) i.v.) also caused neutropenia with an 88% decrease in circulating PMNs after 30 min. When rats were pretreated with F-[OPdChaWR] (0.3 - 10 mg kg(-1) i.v.) 10 min prior to LPS, there was a dose-dependent antagonism of the neutropenia caused by LPS, with up to 69% reversal of neutropenia observed 30 min after LPS administration. These findings suggest that C5a receptor antagonists may have therapeutic potential in the many diseases known to involve either endotoxin or C5a.

The influence of Lys68 in decepeptide agonists of C5a on C5a receptor binding, activation and selectivity.

The potent, conformationally biased C5a agonist peptide YSFKPMPLaR (C5a65-74, Y65, F67, P69, P71, D-Ala73) was used as a template to gain insight into the nature and importance of lysine at position 68 in the peptide-receptor interaction. A panel of YSFKPMPLaR analogs with systematic substitutions for Lys68 was evaluated for C5a receptor (C5aR) binding affinity and activation in two well-characterized assay systems: human polymorphonuclear leukocytes (PMNs) and human fetal artery. In addition, we determined the activity of these new analogs in transfected rat basophilic leukemia (RBL) cells in which the Glu at position 199 of the C5aR (wtGlu199) was replaced by a Gln (C5aR-Gln199) or a Lys (C5aR-Lys199). Our results indicated that Lys68 in YSFKPMPLaR plays an important role in binding the C5aR expressed on PMNs and RBL cells. Furthermore, the data indicated that Lys68 interacted with Glu199 of the C5aR in PMNs and RBL cells. In human fetal artery, however, Lys68 substitutions had little or no effect on activity, which suggested that the receptor conformation may be different in this tissue. Thus, the interaction between Lys68 of the decapeptide agonist and Glu199 of the C5aR may be cell type-specific and may form the molecular basis for tissue-specific responses to C5a agonists.

Small molecular probes for G-protein-coupled C5a receptors: conformationally constrained antagonists derived from the C terminus of the human plasma protein C5a.

Activation of the human complement system of plasma proteins in response to infection or injury produces a 4-helix bundle glycoprotein (74 amino acids) known as C5a. C5a binds to G-protein-coupled receptors on cell surfaces triggering receptor-ligand internalization, signal transduction, and powerful inflammatory responses. Since excessive levels of C5a are associated with autoimmune and chronic inflammatory disorders, inhibitors of receptor activation may have therapeutic potential. We now report solution structures and receptor-binding and antagonist activities for some of the first small molecule antagonists of C5a derived from its hexapeptide C terminus. The antagonist NMe-Phe-Lys-Pro-D-Cha-Trp-D-Arg-CO2H (1) surprisingly shows an unusually well-defined solution structure as determined by 1H NMR spectroscopy. This is one of the smallest acyclic peptides found to possess a defined solution conformation, which can be explained by the constraining role of intramolecular hydrogen bonding. NOE and coupling constant data, slow deuterium exchange, and a low dependence on temperature for the chemical shift of the D-Cha-NH strongly indicate an inverse gamma turn stabilized by a D-Cha-NH. OC-Lys hydrogen bond. Smaller conformational populations are associated with a hydrogen bond between Trp-NH.OC-Lys, defining a type II beta turn distorted by the inverse gamma turn incorporated within it. An excellent correlation between receptor-affinity and antagonist activity is indicated for a limited set of synthetic peptides. Conversion of the C-terminal carboxylate of 1 to an amide decreases antagonist potency 5-fold, but potency is increased up to 10-fold over 1 if the amide bond is made between the C-terminal carboxylate and a Lys/Orn side chain to form a cyclic analogue. The solution structure of cycle 6 also shows gamma and beta turns; however, the latter occurs in a different position, and there are clear conformational changes in 6 vs 1 that result in enhanced activity. These results indicate that potent C5a antagonists can be developed by targeting site 2 alone of the C5a receptor and define a novel pharmacophore for developing powerful receptor probes or drug candidates.

Biologically active conformer of the effector region of human C5a and modulatory effects of N-terminal receptor binding determinants on activity.

A conformationally biased decapeptide agonist of human C5a (C5a55-74Y65,F67,P69,P71,D-Ala73 or YSFKPMPLaR) was used as a functional probe of the C5a receptor (C5aR) in order to understand the conformational features in the C-terminal effector region of C5a that are important for C5aR binding and signal transduction. YSFKPMPLaR was a potent, full agonist of C5a, but at higher concentrations had a superefficacious effect compared to the natural factor. The maximal efficacy of this analogue was 216 +/- 56% that of C5a in stimulating the release of beta-glucuronidase from human neutrophils. C5aR activation and binding curves both occurred in the same concentration range with YSFKPMPLaR, characteristics not observed with natural C5a or more conformationally flexible C-terminal agonists. YSFKPMPLaR was then used as a C-terminal effector template onto which was synthesized various C5aR binding determinants from the N-terminal core domain of the natural factor. In general, the presence of N-terminal binding determinants had little effect on either potency or binding affinity when the C-terminal effector region was presented to the C5aR in this biologically active conformation. However, one peptide, C5a12-20-Ahx-YSFKPMPLaR, expressed a 100-fold increase in affinity for the neutrophil C5aR and a 6-fold increase in potency relative to YSFKPMPLaR. These analyses showed that the peptides used in this study have up to 25% of the potency of C5a in human fetal artery and up to 5% of the activity of C5a in the PMN enzyme release assay.

Molecular adjuvant effects of a conformationally biased agonist of human C5a anaphylatoxin.

A conformationally biased decapeptide agonist of human C5a anaphylatoxin (YSFKPMPLaR) was used as a molecular adjuvant in stimulating Ab responses against peptide epitopes derived from human MUC1 glycoprotein and the human mu and kappa opioid receptors. C57BL6 mice were immunized with the MUC1 epitope (YKQGGFLGL); the C5a agonist (YSFKPMPLaR); YSFKPMPLaR and YKQGGFLGL together, but unconjugated; a C5a-active, MUC1 epitope construct (YKQGGFLGLYSFKPMPLaR); and a C5a-inactive, reversed moiety construct (YSFKPMPLaRYKQGGFLGL). High Ab titers specific for the MUC1 epitope were observed only in mice immunized with the C5a-active epitope construct. Similar results were obtained in BALB/c mice immunized with the C5a-active, MUC1 epitope construct. Abs from the sera of the C57BL6 mice were predominately of the IgG2a, IgG2b, and IgM isotypes and were reactive against human recombinant MUC1 and MUC1 expressed by the Panc-1 M1F.15 pancreatic cell line. When compared with the corresponding KLH-epitope conjugates in C57BL6 mice, the epitope-C5a agonist constructs produced titers of specific IgG Abs of isotypes distinct from those generated by the keyhole limpet hemocyanin-epitope conjugates. Rabbits immunized with a mu opioid receptor epitope-C5a agonist construct (GDLSDPCGNRTNLGGRDSLYSFKPMPLaR) or a kappa opioid receptor epitope-C5a agonist construct (FPGWAEPDSNGSEDAQLYSFKPMPLaR) generated high titer, epitope-specific Ab responses. Ab titers generated in response to the opioid epitope-C5a agonist constructs were comparable to those generated by the opioid KLH-epitope conjugates. The results of this study are discussed in terms of possible mechanisms by which the conformationally biased C5a agonist serves as a molecular adjuvant.

Conformationally biased analogs of human C5a mediate changes in vascular permeability.

A panel of conformationally constrained, decapeptide agonists corresponding to the C-terminal "effector" region of human C5a (C5a65-74 or ISHKDMQLGR) was evaluated for the ability to increase vascular permeability. One constrained analog, acyl-YSFKPMPLaR, expressed between 2 and 10% of full C5a activity in increasing vascular permeability, as measured by the extravasation of Evans blue dye in guinea pig skin. This analog was at least 10-fold more potent than its unconstrained sister analog C5a65-74465, F67++ (YSFKDMQLGR), which was used as an internal standard in these assays. Neither acyl-YSFKPMPLaR nor YSFKDMQLGR changed the transvascular equillibrium of an electrolyte, 86Rb, at the peptide injection site. However, both peptides effected a significant increase in the extravasation of two macromolecules, 125I-labeled bovine serum albumin and 131I-labeled monoclonal antibody BL-3. The extravasation of Evans blue dye mediated by 0.03 to 0.1 nmol of acyl-YSFKPMPLaR was nearly abolished by 1 to 10 nmol of the antihistamine diphenhydramine. For YSFKDMQLGR, however, the sensitivity toward diphenhydramine was observed only at low concentrations of the peptide (1 nmol). When incubated in human and mouse sera, acyl-YSFKPMPLaR was shown to be stable toward the actions of serum carboxypeptidases. However, the unconstrained analog YSFKDMQLGR was rapidly converted to the des-Arg form under the same conditions. Taken together, these results support a growing body of evidence that unique topochemical features expressed in conformationally constrained agonist analogs of C5a contribute favorably to their ability to modulate vascular permeability and to their stability in serum.

The effect of C5a and U46619 on the isolated, perfused human placental lobule: development of a method for the online estimation of tissue fluid accumulation.

A method for the automatic and simultaneous determination of perfusion pressure and fluid accumulation in the isolated, perfused human placental lobule is described. We demonstrated that the inflammatory mediator, C5a, a C5a agonist analogue peptide, and the thromboxane mimetic U46619 caused increased fetal perfusion pressure and increased tissue weight when administered via the fetal arterial circulation. Occlusion of the fetal venous effluent tubing caused significantly greater increases in tissue weight than the pharmacological agents. Detectable increases in tissue weight occurred within 47 +/- 3 sec (n = 21) following pressure increases caused by the pharmacological agents. In each case, the increase in tissue weight was accompanied by an increased permeability of the materno-fetal barrier, shown by the transfer of Evans blue albumin from the fetal circulation to the maternal compartment.

Decapeptide agonists of human C5a: the relationship between conformation and neutrophil response.

A series of decapeptide analogues corresponding to the C-terminal region of the human C5a anaphylatoxin (C5a65-74) was synthesized with residue substitutions to restrict conformational flexibility in the C-terminal region (residues 71-74). These analogues behaved as full agonists of natural C5a in their ability to induce shape change (polarization) and the release of enzyme (beta-glucuronidase) from human neutrophils (PMNs). There was a significant pharmacological correlation between the polarization and enzyme-release assays, suggesting similarities in PMN responsiveness toward these constrained peptides. Good correlations were also observed between these two PMN responses and spasmogenic activity (smooth muscle contraction of human fetal artery). A structure-function analysis for PMN polarization and enzyme release led to the identification of the following preferred backbone conformations: a twisted, helix-like conformation for residues 65-69, an extended conformation for residues 70-71, and a beta-turn of type V for residues (71)72-74. The existence of a C-terminal, type V beta-turn is supported by the NOE (nuclear Overhauser effect) results of two peptides from this series. These conformational features are reminiscent of those that were shown to correlate with the expression of spasmogenic and platelet aggregatory activities in an earlier investigation (Sanderson, S.D.; et al. J. Med. Chem. 1994, 37, 3171). These results suggest that PMNs and the cells responsible for smooth muscle contraction possess C5a receptors that respond to similar topochemical features presented by the agonist peptide ligand.

Reversibility of tachyphylaxis to C5A in guinea pig tissues, perfused human placental lobule, and umbilical artery.

The spasmogenic effect of C5a is mediated by histamine and/or eicosanoids. Tachyphylaxis to this effect of C5a occurs rapidly, but the spasmogenic effects of C5a on a guinea pig lung parenchymal strips, field-stimulated ventricular papillary muscle, and human umbilical artery were completely restored by a 1-h period of drug-free rest, whereas that of guinea pig ileum was not. Perfusion of the isolated human placental lobule with C5a caused a transient pressor response that was largely abolished by indomethacin (5 microM), indicating mediation by cyclooxygenase metabolites. This pressor response to C5a was also completely restored following a 1-h rest period. The results show that tissue rest reverses tachyphylaxis to the spasmogenic effects of C5a in tissues where the response is mediated by cyclooxygenase metabolites. Where the response is mediated by histamine released by mast cells, restoration does not occur, presumably because of the catastrophic nature of mast cell degranulation. Histamine released in guinea pig papillary muscle by C5a may be from non-mast-cell sources.

Decapeptide agonists of human C5a: the relationship between conformation and spasmogenic and platelet aggregatory activities.

A series of decapeptide analogues corresponding to the C-terminal region of human C5a anaphylatoxin (C5a65-74) was synthesized with residue substitutions to restrict conformational flexibility in the C-terminus. These conformationally constrained peptides behaved as agonists of C5a in spasmogenic assays (smooth muscle contraction in human fetal artery, guinea pig ileum, and guinea pig lung parenchyma) as well as guinea pig platelet aggregation. There were significant correlations in the potencies of these peptides between the various assays. A structure-function analysis led to the identification of a preferred backbone conformation that correlated with the expression of these biological responses. These backbone structural motifs were consistent with a helix-like conformation for residues 65-69, an elongated structure for residues 70-71, and a beta-turn of either type II or type V for residues (71)72-74. The most potent of these agonists expressed almost 5% of the potency of natural C5a.

Urine composition in normal subjects after oral ingestion of oxalate-rich foods.

1. Urinary composition was studied in nine healthy adults on unrestricted diet and low-oxalate diet with and without individual oxalate-rich foods. 2. Urine oxalate was constant on the low-oxalate and constant high-oxalate diets and only fluctuated greatly on unrestricted diet. 3. Urine oxalate was mainly due to dietary oxalate which accounts for up to two-thirds of urinary oxalate. 4. Urine oxalate was unaffected by urine volume. 5. Varying percentages of dietary oxalate were absorbed depending on the nature of the foodstuff. 6. Although tea was the main source of dietary oxalate in some people it, like strawberries, did not represent a real risk factor. Chocolates, peanuts, beetroot, rhubarb and spinach were considered as high-risk foods. 7. Calcium oxalate crystalluria at 4 degrees C was increased significantly when the oxalate-rich foods were taken. When urine was examined at 37 degrees C no increase in crystalluria was found.