Effect of hawthorn (Crataegus oxycantha) crude extract and chromatographic fractions on multiple activities in a cultured cardiomyocyte assay.

Extracts of hawthorn (Crataegus oxycantha) have become popular herbal supplements for their well-recognized cardiotonic effects. Many commercial preparations have been used successfully in the treatment of congestive heart failure, although the active principles within these extracts have yet to be conclusively identified. Several hawthorn preparations were studied and found to have negative chronotropic effects in a cultured neonatal murine cardiomyocyte assay using unpaced cells. As compared to conventional cardiac drugs (i.e., epinephrine, milrinone, ouabain, or propranolol), hawthorn extract has a unique activity profile. Hawthorn extract appears to be anti-arrhythmic and capable of inducing rhythmicity in quiescent cardiomyocytes. Hawthorn extract does not cause beta-adrenergic receptor blockade at concentrations which cause negative chronotropic effects. Commercial hawthorn preparations, extracts prepared from dried leaves and those made from dried berries have similar chronotropic activities. When crude extracts are separated using size-exclusion chromatography, several fractions retain multiple cardiac activities. Assays with chromatographic fractions reveal that multiple dissimilar cardioactive components may exist within the extract, making the identification of individual active constituents more challenging.

Protein kinase C-promoted inhibition of Galpha(11)-stimulated phospholipase C-beta activity.

The effects of protein kinase C (PKC) activation on inositol lipid signaling were examined. Using the turkey erythrocyte model of receptor-regulated phosphoinositide hydrolysis, we developed a membrane reconstitution assay to study directly the effects of activation of PKC on the activities of Galpha(11), independent of potential effects on the receptor or on PLC-beta. Membranes isolated from erythrocytes pretreated with 4beta-phorbol-12beta-myristate-13alpha-acetate (PMA) exhibited a decreased capacity for Galpha(11)-mediated activation of purified, reconstituted PLC-beta1. This inhibitory effect was dependent on both the time and concentration of PMA incubation and occurred as a decrease in the efficacy of GTPgammaS for activation of PLC-beta1, both in the presence and absence of agonist; no change in the apparent affinity for the guanine nucleotide occurred. Similar inhibitory effects were observed after treatment with the PKC activator phorbol-12,13-dibutyrate but not after treatment with an inactive phorbol ester. The inhibitory effects of PMA were prevented by coaddition of the PKC inhibitor bisindolylmaleimide. Although the effects of PKC could be localized to the membrane, no phosphorylation of Galpha(11) occurred either in vitro in the presence of purified PKC or in intact erythrocytes after PMA treatment. These results support the hypothesis that a signaling protein other than Galpha(11) is the target for PKC and that PKC-promoted phosphorylation of this protein results in a phosphorylation-dependent suppression of Galpha(11)-mediated PLC-beta1 activation.

Phosphorylation by protein kinase C decreases catalytic activity of avian phospholipase C-beta.

The potential role of protein kinase C (PKC)-promoted phosphorylation has been examined in the G-protein-regulated inositol lipid signalling pathway. Incubation of [32P]Pi-labelled turkey erythrocytes with either the P2Y1 receptor agonist 2-methylthioadenosine triphosphate (2MeSATP) or with PMA resulted in a marked increase in incorporation of 32P into the G-protein-activated phospholipase C PLC-betaT. Purified PLC-betaT also was phosphorylated by PKC in vitro to a stoichiometry (mean+/-S. E.M.) of 1.06+/-0.2 mol of phosphate/mol of PLC-betaT. Phosphorylation by PKC was isoenzyme-specific because, under identical conditions, mammalian PLC-beta2 also was phosphorylated to a stoichiometry near unity, whereas mammalian PLC-beta1 was not phosphorylated by PKC. The effects of PKC-promoted phosphorylation on enzyme activity were assessed by reconstituting purified PLC-betaT with turkey erythrocyte membranes devoid of endogenous PLC activity. Phosphorylation resulted in a decrease in basal activity, AlF4(-)-stimulated activity, and activity stimulated by 2MeSATP plus guanosine 5'-[gamma-thio]triphosphate in the reconstituted membranes. The decreases in enzyme activities were proportional to the extent of PKC-promoted phosphorylation. Catalytic activity assessed by using mixed detergent/phospholipid micelles also was decreased by up to 60% by phosphorylation. The effect of phosphorylation on Gqalpha-stimulated PLC-betaT in reconstitution experiments with purified proteins was not greater than that observed on basal activity alone. Taken together, these results illustrate that PKC phosphorylates PLC-betaT in vivo and to a physiologically relevant stoichiometry in vitro. Phosphorylation is accompanied by a concomitant loss of enzyme activity, reflected as a decrease in overall catalytic activity rather than as a specific modification of G-protein-regulated activity.

Purification and G protein subunit regulation of a phospholipase C-beta from Xenopus laevis oocytes.

Xenopus oocytes exhibit both pertussis toxin-sensitive and -insensitive inositol lipid signaling responses to G protein-coupled receptor activation. The G protein subunits Galphai, Galphao, Galphaq, Galphas, and Gbetagamma all have been proposed to function as activators of phospholipase C in oocytes. Ma et al. (Ma, H.-W., Blitzer, R. D., Healy, E. C., Premont, R. T., Landau, E. M., and Iyengar, R. J. Biol. Chem. 268, 19915-19918) cloned a Xenopus phospholipase C (PLC-betaX) that exhibits homology to the PLC-beta class of isoenzymes. Although this enzyme was proposed to function as a signaling protein in the pertussis toxin-sensitive inositol lipid signaling pathway of oocytes, its regulation by G protein subunits has not been directly assessed. As such we have utilized baculovirus-promoted overexpression of PLC-betaX in Sf9 insect cells and have purified a recombinant 150-kDa isoenzyme. PLC-betaX catalyzes hydrolysis of phosphatidylinositol(4,5)bisphosphate and phosphatidylinositol(4)monophosphate, and reaction velocity is dependent on Ca2+. Recombinant PLC-betaX was activated by both Galphaq and Gbetagamma. PLC-betaX exhibited a higher apparent affinity for Galphaq than Gbetagamma, and Galphaq was more efficacious than Gbetagamma at lower concentrations of PLC-betaX. Relative to other PLC-beta isoenzymes, PLC-betaX was less sensitive to stimulation by Galphaq than PLC-beta1 but similar to PLC-beta2 and PLC-betaT. PLC-betaX was more sensitive to stimulation by Gbetagamma than PLC-beta1 but less sensitive than PLC-beta2 and PLC-betaT. In contrast PLC-betaX was not activated by the pertussis toxin substrate G proteins Galphai1, Galphai2, Galphai3, or Galphao. These results are consistent with the idea that PLC-betaX is regulated by alpha-subunits of the Gq family and by Gbetagamma and do not support the idea that alpha-subunits of pertussis toxin-sensitive G proteins are directly involved in regulation of this protein.

A guanine nucleotide-independent inwardly rectifying cation permeability is associated with P2Y1 receptor expression in Xenopus oocytes.

The functional properties of the G protein-coupled P2Y1 receptor were investigated in Xenopus oocytes. Incubation of oocytes expressing either the human or turkey P2Y1 receptor with adenine nucleotide agonists resulted in an increase in Cl- current and activation of a novel cation current with an inwardly rectifying current-voltage relationship. Activation of either the human P2Y2 (P2U-purinergic) or M1 muscarinic receptor expressed in oocytes resulted in an increase in Cl- current similar to that observed in P2Y1 receptor-expressing oocytes but had no effect on cation current. P2 receptor agonists stimulated both the cation current and Cl- current in P2Y1 receptor-expressing oocytes with EC50 values and an order of potency (2-methylthioadenosine diphosphate > 2-methylthioadenosine triphosphate (2MeSATP) > ATP > UTP) that were similar to those previously observed for activation of phospholipase C in 1321N1 human astrocytoma cells stably expressing the human or turkey P2Y1 receptor. The P2Y receptor antagonists suramin and pyridoxal phosphate 6-azophenyl-2'-4'-disulfonic acid both shifted to the right the concentration-response relationship for 2MeSATP for stimulation of oocyte currents. Although injection of oocytes with either GDPbetaS (guanyl-5'-yl thiophosphate) or GTPgammaS (guanosine 5'-3-O-(thio)triphosphate) resulted in loss of adenine nucleotide-promoted Cl- channel activation, neither guanine nucleotide altered the 2MeSATP-stimulated cation current. These data are consistent with the view that activation of the novel cation current by the P2Y1 receptor does not involve a G protein. Tail current analysis of the novel P2Y1 receptor-associated cation conductance revealed that the open channel current-voltage relationship was outwardly rectifying with a reversal potential of -38 mV for the turkey P2Y1 receptor and -36 mV for the human P2Y1 receptor. Replacement of Na+ with K+ ions in the bathing solution produced a shift in reversal potential to near zero mV, but significant outward rectification remained. The cation current was not permeable to either Ca2+ or Ba2+ and exhibited steady-state inactivation at holding potentials below -60 mV. These results indicate that the P2Y1 receptor exhibits both metabotropic properties and a novel G protein-independent ionotropic response when expressed in Xenopus oocytes.

Lack of discrimination by agonists for D2 and D3 dopamine receptors.

The affinities of D3 dopamine receptors for antagonists are similar to those of D2 receptors. D3 receptors have been reported, however, to have affinities nearly 100-fold higher than those of D2 receptors for some agonists, including (+/-)-7-hydroxy-n,n-dipropyl-aminotetralin (7-OH-DPAT) and quinpirole. This has led to the use of these agonists to try to identify functional responses mediated by D3 receptors in vivo. However, D2 receptors exist in multiple states having high and low affinities for agonists. The G protein-coupled state of D2 receptors is believed to be the functional state of these receptors. When receptors were labeled with the D2 receptor antagonist [125I]-(S)-3-iodo-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5,6- dimethoxysalicylamide ([125I]-NCQ-298) under conditions that promote uncoupling of receptors from G proteins, the affinities of D3 receptors were approximately 130-fold higher than those of D2 receptors for 7-OH-DPAT and quinpirole. When receptors were labeled with the D2 receptor agonist [125I]-(R)trans-7-hydroxy-2-[N-propyl-N-(3'-iodo-2'- propenyl)-amino]tetralin ([125I]-7-OH-PIPAT) under conditions that favor interactions of receptors with G proteins, the affinities of D3 receptors were less than sevenfold higher than the affinities of D2 receptors for the same drugs. Similarly, small differences in the affinities of D2 and D3 receptors for other agonists were seen when receptors were labeled with [125I]-7-OH-PIPAT. These data demonstrate that putative D3 receptor-selective agonists also interact with a high-affinity, G protein-coupled state of D2 receptors. The similarities in affinities of the agonist-preferring state of D2 and D3 receptors means that currently available agonists cannot be used to discriminate between behavioral effects mediated by D2 and D3 receptors.

Mechanisms of up-regulation of D2L dopamine receptors by agonists and antagonists in transfected HEK-293 cells.

Mechanisms underlying agonist- and antagonist-induced up-regulation in HEK-293 cells transfected to express D2 dopamine receptors were investigated. We have reported previously that exposure of cells to agonists and antagonists led to an increase in the density of receptors. The time course of up-regulation on exposure to (-)-sulpiride, a D2 dopamine-selective antagonist, was measured. A lag in the effect of antagonists not seen with up-regulation induced by exposure to agonists was observed. Effects of N-propylnorapomorphine were synergistic with those of cyclic AMP analogs, however, synergistic effects between (-)-sulpiride and cyclic AMP analogs were not observed. These findings suggest that separate mechanisms may be involved in agonist- and antagonist-induced up-regulation. Changes in mRNA levels did not appear to account for the increase in receptors after agonist or antagonist treatment. Results of experiments with cycloheximide, a protein-synthesis inhibitor, suggest that increased protein synthesis, and not decreased protein degradation, is responsible for up-regulation by both NPA and (-)-sulpiride. Studies monitoring receptor recovery after treatment with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, an irreversible alkylating agent, suggest that rates of receptor incorporation into membranes are increased after treatment with either an agonist or an antagonist.

Expression of a cloned P2Y purinergic receptor that couples to phospholipase C.

P2Y purinergic receptors previously have been shown to couple either to activation of phospholipase C through a pertussis toxin-insensitive mechanism or to inhibition of adenylyl cyclase through pertussis toxin-sensitive members of the G1 family of G proteins. These and other pharmacological data strongly suggest that multiple P2Y purinergic receptors exist. Webb et al. [FEBS Lett. 324:219-225 (1993)] cloned a cDNA that, when expressed in frog oocytes, displayed the general pharmacological characteristics of a P2Y purinergic receptor but whose second messenger linkage was not resolved. We have now cloned the meleagrid (turkey) homologue of the previously cloned chick P2Y purinergic receptor and have stably expressed it in a heterologous human cell line (1321N1 astrocytoma cells) to establish its signaling properties. The purinergic receptor agonist 2-methylthio-ATP (2MeSATP) stimulated a marked activation of phospholipase C in 1321N1 cells stably expressing the meleagrid receptor. The order of potency of a series of analogues of ATP and ADP for stimulation of phospholipase C by the receptor expressed in 1321N1 cells [2MeSATP = 2-methylthio-ADP > adenosine 5'-O-(2-thio)diphosphate > ADP > 2-chloro-ATP = adenosine 5'-O-(3-thio)triphosphate > or = ATP > adenylyl-imidodiphosphate > UTP] was similar to that observed for P2Y purinergic receptors in turkey erythrocytes and many other tissues and was markedly different from those of the P2U and P2X purinergic receptor subtypes. Stimulation of inositol lipid hydrolysis by P2Y purinergic agonists was not affected by preincubation of cells with pertussis toxin. In contrast to its marked effects on phospholipase C activity, 2MeSATP caused only a small and variable inhibition of cAMP accumulation. Ribonuclease protection analysis of turkey tissues showed that this P2Y purinergic receptor is most highly expressed in blood and brain. Taken together, these results indicate that a phospholipase-C-activating P2Y purinergic receptor has been cloned and stably expressed in 1321N1 astrocytoma cells.

Characterization of [125I](R)-trans-7-hydroxy-2-[N-propyl-N-(3'-iodo-2'-propenyl)amino] tetralin binding to dopamine D3 receptors in rat olfactory tubercle.

[125I](R,S)-trans-7-Hydroxy-2-[N-propyl-N-(3'-iodo-2'-propenyl)- amino]tetralin ([125](R,S)-trans-7-OH-PIPAT) has been shown to bind with high affinity to dopamine D3 receptors expressed in Spodoptera frugiperda cells. No specific binding was seen in Spodoptera frugiperda cells expressing a high density of D2 receptors. It was therefore, suggested that [125I] (R,S)-trans-7-OH-PIPAT selectively labels D3 receptors. In the present study, saturation binding of [125I](R)-trans-7-OH-PIPAT to membranes from rat olfactory tubercle resulted in markedly curvilinear Scatchard plots, suggesting that the radioligand was binding to more than one receptor class or affinity state. [125I] (R)-trans-7-OH-PIPAT bound with high affinity to membranes from human embryonic kidney-293 cells expressing transfected D2 or D3 receptors and to membranes from Chinese hamster ovary cells expressing serotonin1A receptors. Binding of [125I](R)-trans-7-OH-PIPAT to serotonin1A and D2 receptors was decreased or eliminated in the presence of NaCl and/or guanylyl-imidodiphosphate [Gpp(NH)p]. In the presence of Gpp(NH)p and NaCl, a linear Scatchard plot with a Kd value of 0.4 nM and a density of 100 fmol/mg of protein was obtained in membranes from rat olfactory tubercle. Agonists and antagonists inhibited binding of [125I](R)-trans-7-OH-PIPAT with a rank order of potency consistent with an interaction at D3 receptors. These results suggest that, in the presence of Gpp(NH)p and NaCl, [125I](R)-trans-7-OH-PIPAT specifically labels D3 receptors.

Paradoxical regulation of dopamine receptors in transfected 293 cells.

Selective expression of subtypes of receptors in mammalian cell lines permits the study of the regulation of receptors in a homogeneous population of cells growing under controlled conditions. cDNAs encoding the human D2L and D2S receptors were ligated into a eukaryotic expression vector, pRc/CMV. The resulting plasmid, which contains a cytomegalovirus promoter for high expression levels, was used for stable transfection of 293 cells, a human kidney cell line. Expression of D2L and D2S receptors in 293 cells was confirmed by radioligand binding assays with [125I]NCQ 298. The pharmacological properties of the expressed receptors were comparable to those of receptors in rat striatal homogenates and in other transfected cell lines. D2L and D2S receptors were coupled to inhibition of cAMP accumulation in 293 cells. Incubation of 293-D2L cells with agonists resulted in an increase in the density of D2 receptors without a change in the affinity of the receptors for [125I]NCQ 298. This effect was time dependent, with a t1/2 of approximately 6 hr. The dose dependence of up-regulation followed the pharmacological profile expected of a D2 receptor, with an order of potency of N-propylnorapomorphine (NPA) > quinpirole > dopamine. The density of receptors was further increased by incubation of cells with agonist together with forskolin or 8-bromo-cAMP. D2S receptors responded similarly to D2L receptors to treatment with NPA and forskolin. Exposure of 293-D2L cells to the beta-adrenergic receptor agonist isoproterenol did not change the density of D2L receptors. Similarly, NPA had no effect on levels of endogenously expressed beta-adrenergic receptors in 293-D2L cells, as assayed by binding of [125I]iodocyanopindolol. Levels of beta-adrenergic receptors in transfected 293-beta 2 or 293-D2L cells did not increase after exposure to NPA but decreased after exposure to isoproterenol. Cells expressing D2L receptors were incubated with antagonists, including SCH-23390, sulpiride, haloperidol, clozapine, and epidepride, alone or in combination with NPA. Incubation of cells with SCH-23390 had no effect on the density of D2 receptors, and SCH-23390 did not block the effect of NPA. D2-selective antagonists increased the density of receptors. D2L receptor mRNA levels were unchanged during agonist treatment. This suggests a role for translational or post-translational mechanisms in the regulation of D2 receptor levels in transfected cell lines.

Development of polyclonal anti-D2 dopamine receptor antibodies using sequence-specific peptides.

Multiple subtypes of dopamine receptors with similar properties have been described. Ligands that have been shown to interact with a single subtype of receptor do not yet exist. The use of immunologic methods provides an alternative approach to distinguish receptors and receptor isoforms. Synthetic peptides corresponding to portions of the third intracellular loops of the two isoforms of the rat D2 dopamine receptor were used to elicit polyclonal antipeptide antibodies. Peptide D2-244 is unique to the D2L isoform, whereas peptide D2-284 is present in both the D2L and the D2S isoforms. Rabbits were immunized monthly with peptide coupled to keyhole limpet hemocyanin. The immunogenicity of the peptides was established using a solid-phase radioimmunoassay. Both immunogens elicited antipeptide antibodies within 10 weeks of the primary immunization, with titers of at least 1/10(4). An immunoprecipitation assay using receptors in digitonin-solubilized extracts of rat or canine caudate labeled with the high affinity D2 antagonist 125I-NCQ 298 showed that antipeptide antisera could recognize solubilized D2 receptors. At a dilution of 1/1000, antisera to peptide D2-284 quantitatively immunoprecipitated 125I-NCQ 298 binding sites from both rat and canine striatal tissue, whereas antisera against peptide D2-244 immunoprecipitated 40% of the D2 receptors solubilized from rat caudate. The selectivity of the antisera was determined using 293 cells transfected with cDNA encoding the D2L or the D2S isoform of receptor. Antisera to D2-284, at a dilution of 1/1000, were able to quantitatively immunoprecipitate receptor from both 293-D2L and 293-D2S cells. Antisera to D2-244 were specific for the D2L isoform, immunoprecipitating 125I-NCQ 298 binding sites from 293-D2L cells but not from 293-D2S cells. Anti-D2-284 specifically recognized multiple bands of 100 kDa, 68 kDa, and 50 kDa in immunoblots of denatured preparations of rat caudate. Immunohistochemical studies with anti-D2-284 demonstrated the presence of the D2 receptor in several regions of rat brain. Immunostaining was most dense in the striatum, with a lateral to medial gradient and patches of lighter staining. Immunoreactivity was negligible with preimmune serum or peptide-blocked immune serum. Immunoreactive processes were seen in the nucleus accumbens and ventral pallidum, as well as in the hypothalamus. The high affinity binding of agonist to D2 dopamine receptors was disrupted by anti-D2-284 but not anti-D2-244 antisera, implicating the internal region of the third intracellular loop represented by peptide D2-284 as a potential determinant of receptor-guanine nucleotide-binding protein coupling.

Expression and characterization of the rat D3 dopamine receptor: pharmacologic properties and development of antibodies.

A baculovirus expression system provided an enriched source of biologically and immunologically active D3 dopamine receptors. Receptors expressed in Spodoptera frugiperda insect (Sf9) cells at a density of 5 to 15 pmol/mg of protein displayed high affinity for the antagonists, eticlopride, fluphenazine and spiroperidol, and the agonist, N-propylnorapomorphine. The binding of agonists was not sensitive to GTP. Antisera raised against synthetic peptides in the third intracellular loop of the D3 dopamine receptor immunoprecipitated binding sites for (S)-3-[125I]-iodo-2-hydroxy-5,6-dimethoxy-N-[(1-ethyl-2-pyrrolidinyl)- methyl]-benzamide from solubilized extracts of infected Sf9 cells and detergent extracts of rat caudate. These antisera specifically recognized a single band on immunoblots of Sf9 cells infected with recombinant D3 baculovirus. Both the immunoprecipitation and immunoblot reactions were blocked by preincubation of the antisera with the immunization peptide. These results suggest that the D3 receptor protein is expressed in rat brain.

Synthesis and applications of an aldehyde-containing analogue of SCH-23390.

SCH-23390 is a high-affinity antagonist selective for D1 dopamine receptors (Ki = 2.5 nM). It does not contain a functional group that can be conveniently coupled to commercially available resins for affinity chromatography or to prepare photolabels for photoaffinity labeling of receptors. To construct an affinity resin for purification of dopamine D1 receptors, an aldehyde analogue of SCH-23390, (+/-)-7-chloro-8-hydroxy-1-(4'-formylphenyl)-3-methyl-2,3,4,5-tetrahydro -1H- 3-benzazepine (ASCH), was synthesized. 8-Methoxy-1-(4'-bromophenyl)-SCH-23390 was lithiated, formylated, and O-demethylated to form the aldehyde. NMR and IR analyses were performed to characterize the product. Assays were performed with the radioligand [125I]SCH-23982 to define the biological activity of the aldehyde. ASCH displaced [125I]SCH-23982 binding from caudate membranes with a Ki value of 7.1 nM. ASCH has been coupled through the aldehyde group on the phenyl ring to diaminodipropylamine-agarose for affinity chromatography. After solubilization of caudate membranes in 1% digitonin, the affinity resin retained binding sites for [125I]SCH-23982 that were eluted with 10 mM SCH-23390. The aldehyde was also covalently coupled to biotin hydrazide for fluorescence labeling of dopamine D1 receptors. The biotin-conjugated aldehyde of SCH-23390 displaced [125I]SCH-23982 binding from caudate membranes with a Ki value of 9.3 nM.

Role of excitatory amino acids in rat vagal and sympathetic baroreflexes.

Vagal baroreflexes were studied by measuring the atropine-sensitive cardioinhibition produced by raising arterial pressure with phenylephrine in anesthetized rats pretreated with the beta-adrenergic receptor antagonist nadolol. Sympathetic baroreflexes were determined in halothane-anesthetized rats by measuring the inhibition of lumbar sympathetic discharge produced by elevating arterial pressure with gradual aortic constriction. Both reflexes were drastically reduced by bilateral injections of 2.2 nmol of the glutamate receptor antagonist kynurenic acid (KYN) into either the nucleus of the solitary tract (NTS) or the ventrolateral medulla between 0 and 1 mm posterior to the level of the obex. Injections of KYN elsewhere in the medulla were generally ineffective and injections of 8-OH kynurenate (an inactive analog) into the ventrolateral medulla or NTS were also without effect. KYN injections (2.2 nmol) into the intermediate portion of the NTS produced small increases in mean arterial pressure (0-15 mm Hg) and no change in heart rate while injections of similar amounts into the ventrolateral medulla at obex level were followed by large (35-116 mm Hg) increases in pressure and bradycardia. Both types of injections produced a similar degree of blockade of vagal and sympathetic baroreflexes. These results support previous evidence that baroreceptor primary afferents may release a glutamate-like transmitter in the NTS and indicate that a similar type of excitatory transmitter is involved at the level of the ventrolateral medulla in mediating or modulating both vagal and sympathetic baroreflexes. Finally the bradycardia and hypertension produced by blocking amino acid receptors in the ventrolateral medulla appear largely unrelated to the disruption of peripheral baroreceptor inputs.