Biomedical subjects
Publications and source records attributed to P J Worland.
Cyclin dependent kinases (CDKs) along with the complementary cyclins form key regulatory checkpoint controls on the cell cycle. Flavopiridol is a synthetic flavone that shows potent and selective cyclin-dependent kinase inhibitory activity. In this paper, we report modifications of the 3-hydroxy-1-methylpiperidinyl (D ring) of flavopiridol and their effect on CDK inhibitory activity.
Flavopiridol is a novel flavonoid that induces cell cycle arrest at different stages of the cell cycle because of the inhibition of cyclin-dependent kinases (cdks). In previous studies from our laboratory, (B. A. Carlson et al., Cancer Res., 56: 2973-2978, 1996), we observed that exposure of the MCF-7 breast carcinoma cell line to flavopiridol resulted in G1-S arrest, which was associated with the loss of cdk4 and cdk2 activity by 24 h of exposure. Along with this inhibition, flavopiridol decreased total cyclin-D protein levels in this cell line. In this work, we demonstrate that using isoform-specific antibodies, flavopiridol induces an early (by 6 h) decrease in cyclin D1 protein levels. This decline is followed by a decline in cyclin D3 with no effect on cyclin D2 or cyclin E levels by 10 h. Furthermore, at early time points (up to 8 h), the activity of cdk4 and the expression of endogenous phosphorylated retinoblastoma species from intact cells exposed to flavopiridol are unchanged. Thus, the decline in cdk4 activity and the induction of retinoblastoma hypophosphorylation follows cyclin D1 decline. Turnover studies demonstrate that the half-life of cyclin D1 (approximately 30 min) is not shortened in flavopiridol-exposed cells, and that the turnover of cdk4-bound cyclin D1 is unaltered. However, steady-state levels of cyclin D1 mRNA display a significant decrease by 4 h of flavopiridol treatment, with total disappearance by 8 h. This mRNA decline is not abrogated by the presence of cycloheximide. Furthermore, we have found that flavopiridol specifically represses the activity of the full-length cyclin D1 promoter linked to a luciferase reporter gene. In summary, we have found that the flavopiridol-induced decline in cyclin D1 is an early event, specific and, at least in part, due to the transcriptional repression of the cyclin D1 promoter. These results extend our understanding of flavopiridol's action to include regulation of cyclin D1 transcription.
BACKGROUND: Arrest of the cell cycle in G2 phase following DNA damage helps protect cell viability by allowing time for DNA repair before entry into mitosis (M phase). Abrogation of G2 arrest sensitizes cells to the effects of DNA-damaging agents. UCN-01 (7-hydroxystaurosporine), a protein kinase C inhibitor that may block G2 checkpoint regulation, has been reported to enhance the cytotoxicity of mitomycin C, a known DNA-damaging agent. PURPOSE: We studied the effect of UCN-01 on G2 checkpoint control in human lymphoma CA46 cells, whose sensitivity to various DNA-damaging agents and G2 response to DNA damage have been characterized. We also assessed the ability of UCN-01 to enhance the cytotoxicity of gamma irradiation in CA46 cells and human colon carcinoma HT-29 cells, both of which are mutant for p53 function. The influence of p53 function on UCN-01-mediated abrogation of the G2 checkpoint and enhancement of DNA-damaging agent cytotoxicity was studied in transfected human breast carcinoma MCF-7 cells that either expressed or did not express the human papillomavirus type-16 E6 protein. MCF-7 cells have normal p53 function, and the E6 protein binds p53 protein and promotes its destruction. METHODS: The effect of UCN-01 on cell cycle arrest induced by gamma irradiation was studied in CA46 cells and in transfected MCF-7 cells by use of flow cytometry. A histone H1 phosphorylation assay was employed to measure cyclin B1/Cdc2 kinase activity in extracts derived from irradiated and nonirradiated CA46 cells that had been either treated or not treated with UCN-01; the phosphorylation status of Cdc2 kinase protein in the same extracts was determined by use of western blotting. The effect of UCN-01 on the cytotoxicity of gamma irradiation in CA46 and HT-29 cells was determined by use of MTT (thiazolyl blue) and clonogenic (colony-forming) assays, respectively; a clonogenic assay was also used to measure the effect of UCN-01 on the cytotoxicity of cisplatin in transfected and nontransfected MCF-7 cells. RESULTS: G2 arrest induced in CA46 cells by gamma irradiation was minibited by treatment with UCN-01 in a dose-dependent manner; arrest in G2 was completely abrogated by exposure to 300 nM UCN-01. Biochemical markers indicative of the G2/M transition, including the activation of cyclin B1/Cdc2 kinase and the suppression of Cdc2 threonine-14 and tyrosine-15 phosphorylation, were detected in irradiated cells treated with UCN-01. UCN-01 enhanced the cytotoxicity of gamma irradiation in CA46 and HT-29 cells. MCF-7 cells with functional p53 protein were more resistant to G2 checkpoint abrogation by UCN-01 than MCF-7 cells with disrupted p53 function. UCN-01 markedly enhanced the cell-killing activity of cisplatin in MCF-7 cells defective for p53 function. CONCLUSIONS AND IMPLICATIONS: UCN-01 is a potent abrogator of G2 checkpoint control in cancer cells with disrupted p53 function. UCN-01 might be capable of enhancing the effectiveness of DNA-damaging agents in the treatment of tumors with cells lacking normal p53 function.
Flavopiridol (L86-8275), a N-methylpiperidinyl, chlorophenyl flavone, can inhibit cell cycle progression in either G1 or G2 and is a potent cyclin-dependent kinase (CDK) 1 inhibitor. In this study, we used MCF-7 breast carcinoma cells that are wild type for p53 and pRb positive and contain CDK4-cyclin D1 and MDA-MB-468 breast carcinoma cells that are mutant p53, pRb negative, and lack CDK4-cyclin D1 to investigate the G1 arrest produced by Flavopiridol. Recombinant CDK4-cyclin D1 was inhibited potently by Flavopiridol (Kiapp, 65 nM), competitive with respect to ATP. Surprisingly, CDK4 immunoprecipitates derived from Flavopiridol-treated MCF-7 cells (3 h, 300 nM Flavonolpiridol) had an approximately 3-fold increased kinase activity compared with untreated cells. Cyclin D and CDK4 levels were not different at 3 hr, but cyclin D levels and CDK4 kinase activity decreased thereafter. The phosphorylation state of pRb was shifted from hypercoincident to hypocoincident with the development of G1 arrest. Asynchronous MDA-MB-468 cells were inhibited in cell cycle progression at both G1 and G2 by Flavopiridol. Flavopiridol inhibited the in vitro kinase activity of CDK2 using an immune complex kinase assay (IC50, 100 nM at 400 microM ATP). Immunoprecipitated CDK2 kinase activity from either MCF-7 or MDA-MB-468 cells exposed to Flavopiridol (300 nM) for increasing time showed an initial increased activity (approximately 1.5-fold at 3 h) compared with untreated cells, followed by a loss of kinase activity to immeasurable levels by 24 h. This increased immunoprecipitated kinase activity was dependent on the Flavopiridol concentration added to intact cells and was associated with a reduction of CDK2 tyrosine phosphorylation. Cyclin E and A levels were not altered to the same extent as cyclin D, and neither CDK4 nor CDK2 levels were changed in response to Flavopiridol. Inhibition of the CDK4 and/or CDK2 kinase activity by Flavopiridol can therefore account for the G1 arrest observed after exposure to Flavopiridol.
The central role of cyclin-dependent kinases (CDKs) in cell cycle regulation makes them a promising target for studying inhibitory molecules that can modify the degree of cell proliferation. The discovery of specific inhibitors of CDKs such as polyhydroxylated flavones has opened the way to investigation and design of antimitotic compounds. A novel flavone, (-)-cis-5,7-dihydroxyphenyl-8-[4-(3-hydroxy-1-methyl)piperidinyl] -4H-1-benzopyran-4-one hydrochloride hemihydrate (L868276), is a potent inhibitor of CDKs. A chlorinated form, flavopiridol, is currently in phase I clinical trials as a drug against breast tumors. We determined the crystal structure of a complex between CDK2 and L868276 at 2.33 angstroms resolution and refined to an Rfactor 20.3%. The aromatic portion of the inhibitor binds to the adenine-binding pocket of CDK2, and the position of the phenyl group of the inhibitor enables the inhibitor to make contacts with the enzyme not observed in the ATP complex structure. The analysis of the position of this phenyl ring not only explains the great differences of kinase inhibition among the flavonoid inhibitors but also explains the specificity of L868276 to inhibit CDK2 and CDC2.
The abundant, cytoplasmic 90-kDa heat-shock protein associates transiently with the Rous sarcoma virus oncogenic protein tyrosine kinase, pp60v-src, directs its cellular trafficking and negatively regulates its kinase activity. Here we report that the serine/threonine phosphatase inhibitor, okadaic acid, destabilized the heat-shock protein 90-pp60v-src chaperone complex in v-src-transfected cells. Concomitant with complex destabilization by okadaic acid, phosphoserine was doubled and phosphothreonine was increased 20-fold in the heat-shock protein 90. Although phosphorylation of the total pool of immunoprecipitable pp60v-src was unchanged, okadaic acid slightly increased phosphoserine and phosphothreonine levels specifically in pp60v-src bound to heat-shock protein 90. The low level of tyrosine phosphorylation in the pp60v-src complexed with heat-shock protein 90 was further decreased by okadaic acid. Interestingly, okadaic acid-stabilized hyperphosphorylation of the heat-shock protein 90-pp60v-src complex lowered the level of pp60v-src in cell membranes, the functional location for pp60v-src. We suggest that serine/threonine phosphorylation of heat-shock protein 90 and/or pp60v-src functions as a regulatory molecular trigger to release pp60v-src from the chaperone complex at the inner surface of cell membranes.
Several guanine nucleotide-binding protein-coupled receptors are known to be rapidly phosphorylated after agonist exposure. In this study we show that the gastrin-releasing peptide receptor (GRP-R) is rapidly phosphorylated in response to agonist exposure. When [32P]orthophosphate-labeled cells were exposed to bombesin, the receptor was maximally phosphorylated on serine and threonine residues within 1 min. Although addition of 12-O-tetradecanoylphorbol 13-acetate also resulted in phosphorylation of the GRP-R, elimination of protein kinase C activity using the inhibitor 7-hydroxystaurosporine did not prevent bombesin-induced GRP-R phosphorylation. We conclude that a kinase other than protein kinase C is principally responsible for the rapid, agonist-induced phosphorylation of the GRP-R.
7-Hydroxystaurosporine (UCN-01) is a potent inhibitor of protein kinase C (PKC) isozymes alpha, beta, and gamma [Seynaeve et al., Mol. Pharmacol, 45: 1207-1214, 1994] that also has antitumor effects in vivo. To determine whether inhibition of PKC can be related to inhibition of cell growth with induction of apoptosis, we compared the effects of UCN-01 to those of the highly selective bisindolylmaleimide PKC antagonist GF 109203X in leukemic T-cell lines. Both compounds potently inhibited PKC activity when added to T-cell membrane preparations and reversed phorbol ester-induced c-fos gene expression in intact cells. However, whereas UCN-01 potently inhibited growth of Jurkat, Molt-3, Molt-4, and Hut-78 cells (IC50 = 20-65 nM, irreversible after 24 h of exposure), GF 109203X had IC50s for cell growth of 3.6-5.0 muM. Less than 3 h after addition, UCN-01 but not GF 109203X-treated cells displayed loss of cells with G2-M DNA content, appearance of a hypodiploid DNA fraction, and evidence of internucleosomal DNA fragmentation. Six h after treatment, cells appeared to accumulate with S-phase DNA content. These effects correlated with selective UCN-01 but not GF 109203X-induced decrease in total and tyrosine phosphorylation of cyclin-dependent kinases (cdks) 1 and 2, and with increases in the histone H1 kinase activities of cdk1 and cdk2. UCN-01 was relatively less potent in inhibition of properly activated cdk1 and cdk2 when added in vitro to H1 kinase assays (IC50 = 1000 and 600 nM, respectively). We conclude that inhibition of PKC alone is not sufficient to account for the actions of UCN-01 and are led to the hypothesis that inappropriate cdk activation either correlates with or actually mediates cell growth inhibition with apoptosis in T lymphoblasts exposed to UCN-01.
L86-8275 [(-) cis-5,7-dihydroxy-2-(2-chlorophenyl)-8[4-(3-hydroxy-1- methyl)-piperidinyl]-4H-benzopyran-4-one] directly inhibits immunoprecipitated Cdc2 kinase activity from G2/M synchronized MDA-MB-468 breast carcinoma cells and is at least 250-fold more potent than either quercetin or genistein. Purified sea-star Cdc2 kinase (IC50 = 0.5 microM) was inhibited with a similar potency to immunoprecipitated Cdc2 kinase from MDA-MB-468 cells (IC50 = 0.4 microM). This inhibition was competitive with respect to ATP (KiATP = 0.041 microM) and noncompetitive with respect to a synthetic peptide substrate, CDK1S1 (AAKAKKTPKKAKK-CONH2, KiCDK1S1 = 0.14 microM). These data suggest L86-8275 as a lead structure for the development of inhibitors of the cyclin-dependent kinases.
UCN-01 (7-hydroxystaurosporine) has been demonstrated to be a potent inhibitor of tumor cell growth both in cell culture and with in vivo xenograft models. The ability of UCN-01 to inhibit the kinase activity of recombinant protein kinase C (PKC) isozymes alpha, beta, gamma, delta, epsilon, and zeta was characterized using an in vitro kinase assay. Two distinct groups of isozymes could be defined on the basis of relative potency of kinase inhibition. UCN-01 was 15-20-fold more potent for inhibition of the Ca(2+)-dependent isozymes, compared with the Ca(2+)-independent isozymes. In contrast, UCN-02 (the diastereomer of UCN-01) and staurosporine exhibited less ability to discriminate between Ca(2+)-dependent and -independent isozymes. PKC-zeta was not inhibited by UCN-01, UCN-02, or staurosporine. IC50 values for UCN-01 inhibition of the Ca(2+)-dependent PKC-alpha, -beta, and -gamma were 29, 34, and 30 nM, respectively, and for the Ca(2+)-independent PKC-delta and -epsilon were 530 and 590 nM, respectively. IC50 values for staurosporine inhibition of the isozymes alpha, beta, and gamma were 58, 65, and 49 nM, respectively, and for the isozymes delta and epsilon were 325 and 160 nM, respectively. UCN-02 was significantly less potent for the inhibition of PKC-alpha, -beta, -gamma, -delta, and -epsilon (IC50 values of 530, 700, 385, 2800, and 1200 nM, respectively). An analysis of the inhibition by UCN-01 and staurosporine of the kinase activity of PKC-alpha and -delta indicated mixed inhibition kinetics. Increasing the ATP concentration resulted in decreased potency, as shown by increased IC50 values. In contrast, increasing the peptide substrate concentration resulted in increased potency, as shown by decreased IC50 values. Increasing concentrations of myelin basic protein as a PKC-alpha or -delta substrate also caused increased potency of inhibition by UCN-01. Because of the competitive nature of inhibition with respect to ATP and the uncompetitive nature with respect to substrate, the concentrations of these substrates can have dramatically different effects on the degree of inhibition observed. These data also suggest that UCN-01 may be an important tool for the dissection of PKC isozyme contributions to signal transduction pathways.
The flavone L86-8275 [(-)cis-5,7-dihydroxy-2-(2-chlorophenyl)-8-[4-(3-hydroxy-1-methyl)- piperidinyl]-4H-1-benzopyran-4-one] delayed the progression of aphidicolin-synchronized MDA-468 breast carcinoma cells through S phase and prevented progression through G2. L86-8275 prevented the G2-related increase in histone H1 kinase activity mediated by cyclin-dependent kinase-1 (p34cdc2 kinase). L86-8275 inhibited [32P]orthophosphate labeling of p34cdc2 threonine and tyrosine residues and decreased the phosphotyrosine content of p34cdc2. Diminution of p34cdc2 phosphotyrosine appeared selective, as a general depletion of cellular phosphotyrosine was not observed. The mass of p34cdc2 in L86-8275-exposed cells was not decreased during the period over which these effects occurred. [35S]Methionine labeling of p34cdc2 or other cellular proteins was not inhibited at concentrations that were effective for complete cellular growth inhibition. We hypothesize that L86-8275 interferes with the normal cell cycle-dependent phosphorylation of p34cdc2, resulting in decreased kinase activity and cell cycle arrest.
UCN-01 is a derivative of staurosporine, initially developed as a potentially selective inhibitor of the Ca(2+)- and phospholipid-dependent protein kinase C, but with the capacity to inhibit a number of tyrosine and serine/threonine kinases. UCN-01 inhibits the growth of 5 breast carcinoma cell lines with a 50% inhibitory concentration range of 30-100 nM during 6 days of continuous exposure. In MCF-7, MDA-MB453, and SK-BR-3 cells, UCN-01 is 5-fold more potent in growth inhibition than its diastereomer UCN-02, but the 2 compounds are equipotent in the inhibition of MDA-MB468 and H85787 cell growth. A differential sensitivity to a 24-h period of exposure to UCN-01 followed by drug removal and growth for 5 subsequent days was observed. The rank order for persistent inhibition of cells by UCN-01 was MCF-7, MDA-MB453 >> SK-BR-3 > H85787 > MDA-MB468. MCF-7 and MDA-MB453 cells did not resume proliferation within the 5 days after brief exposure to UCN-01. In contrast, MDA-MB468 and H85787 cells showed no net growth inhibition after a 24-h pulse of UCN-01, followed by 5 more days of growth in drug-free medium. In MDA-MB468 cells, 150 nM UCN-01 retards but does not prevent cell cycle progression through S phase, but the cells are clearly blocked from exit of G1 and entry into S. Progression through S phase is completely inhibited by 600 nM UCN-01. The development of a G1 to S block by UCN-01 in MDA-MB468 cells occurs in conjunction with inhibition of [32P]orthophosphate labeling and decreased phosphotyrosine mass of discrete cellular phosphoproteins.
Clonal cell lines were derived from rat liver epithelial cells following their transformation with either v-raf or v-raf/v-myc. Cells transformed with v-raf alone showed reduced tumor incidence and tumor growth rates when implanted into nude mice, compared to cells also expressing the v-myc oncogene. A series of additional clones isolated from a tumor obtained following inoculation of an athymic nude mouse with the v-raf-transformed rat liver epithelial cells displayed an intermediate range of tumor aggressiveness. These findings indicate that unknown genotypic and/or phenotypic changes occur during tumor formation in vivo, which are required in addition to raf activation for complete expression of the malignant phenotype. This in vitro model of tumor progression was used to examine alterations in the expression of genes related to the growth control of liver epithelial cells, which may be involved in the malignant conversion of the preneoplastic cells. A close association was observed between the increased level of expression of the transforming growth factors alpha and beta 1, the decreased expression of extracellular matrix proteins fibronectin and collagen I, and the tumor aggressiveness (latency/growth rate), suggesting a causal role for these factors in the progression of v-raf-transformed rat liver epithelial cells to the fully malignant phenotype.
A series of clonal cell lines were derived from rat liver epithelial cells after being infected with a defective retrovirus containing either v-raf (3611-MSV) or v-raf/v-myc(J2) together with a helper virus. These clones exhibited a different morphology from the regular cuboid shape of the control cells, infected only with the helper virus. All of the infected cell lines contained at least one full length copy of appropriate proviral DNA and expressed comparable levels of v-raf mRNA, although only the cells transformed with the v-raf/v-myc combination were capable of anchorage-independent growth in soft agar. All of the clones except the controls formed tumors in nude mice but with markedly different latency periods and growth rates. Thus, these cell lines represent an in vitro model for tumor progression. Two-dimensional polyacrylamide electrophoresis was used to investigate changes in cellular protein expression related to malignant conversion. The expression of three proteins of pI/Mr x 10(-3) 5.9-7.2/205 (RP1), 6.5-7.5/160 (RP2) and 4.0/85 (RP3) consistently matched the transformed phenotype. In particular the expression of RP1 and RP2 correlated with the relative tumorigenicity of the cell lines. Rat liver epithelial cell lines transformed by other protocols that did not involve v-raf also showed downregulation of these three polypeptides. Crude fractionation studies determined RP1 to be soluble and RP2 and RP3 to be membrane associated. RP2 was shown to be a glycoprotein containing mannose and galactose residues. These three proteins are consistent markers for the tumorigenic potential of rat liver epithelial cells.
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The presence of a unique opal suppressor seryl-tRNA in higher vertebrates which is converted to phosphoseryl-tRNA has been known for several years, but its function has been uncertain (see Hatfield, D. (1985) Trends Biochem. Sci. 10, 201-204 for review). In the present study, we demonstrate that this tRNA species also occurs in vivo as selenocysteyl-tRNA(Ser) suggesting that it functions both as a carrier molecule upon which selenocysteine is synthesized and as a direct selenocysteine donor to a growing polypeptide chain in response to specific UGA codons. [75Se]Seleno[3H]cysteyl-tRNA(Ser) formed by administering 75Se and [3H]serine to rat mammary tumor cells (TMT-081-MS) in culture was isolated from the cell extract. The amino acid attached to the tRNA was identified as selenocysteine following its deacylation and reaction with iodoacetate and 3-bromopropionate. The resulting alkyl derivatives co-chromatographed on an amino acid analyzer with authentic carboxymethylselenocysteine and carboxyethylselenocysteine. Seryl-tRNA(Ser) and phosphoseryl-tRNA(Ser) (Hatfield, D., Diamond, A., and Dudock, B. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 6215-6219), which co-migrate on a reverse phase chromatographic column with selenocysteyl-tRNA(Ser), were also identified in extracts of TMT-018-MS cells. Hence, we propose that a metabolic pathway for selenocysteine synthesis in mammalian cells is the conversion of seryl-tRNA(Ser) via phosphoseryl-tRNA(Ser) to selenocysteyl-tRNA(Ser). In a ribosomal binding assay selenocysteyl-tRNA(Ser) recognizes UGA but not any of the serine codons. Selenocysteyl-tRNA(Ser) is deacylated more readily than seryl-tRNA(Ser) (i.e. 58% deacylation during 15 min at pH 8.0 and 37 degrees C as compared to 41%).
The polypeptide patterns of MCF-7 human breast cancer cells (MCF-7gpt) and a stably v-H-ras-transfected subclone (MCF-7ras) have been analyzed following estradiol treatment. Since both estradiol and v-H-ras transfection increase tumorigenicity of MCF-7 cells, this study was designed to ascertain if specific changes in polypeptides were common in both treatments. Separation of cellular and secreted polypeptides was accomplished by 2-dimensional polyacrylamide gel electrophoresis, and the consequent patterns were analyzed with computer assistance. Estradiol treatment of the MCF-7gpt cells reduced the number of differences found in the polypeptide patterns between MCF-7gpt and MCF-7ras. Twelve cellular polypeptides were consistently modulated by either estradiol or v-H-ras, with four polypeptides clearly affected in the same way by both treatments. Polypeptides Gchc-0845 (Mr 54,000, pI 6.9) and Gchc-0902 (Mr 52,000, pI 6.3) were suppressed by estradiol and v-H-ras, while Gchc-1240 (Mr 34,000, pI 4.4) and Gchc-1396 (Mr 23,000, pI 5.3) were induced by estradiol and v-H-ras. Sixteen secreted polypeptides were altered by at least 2-fold subsequent to estradiol treatment or v-H-ras transfection. Transfection with v-H-ras had a greater effect than estradiol, stimulating the secretion of eight polypeptides and suppressing the secretion of seven polypeptides compared to estradiol which increased secretion of five polypeptides and decreased secretion of an additional three polypeptides, respectively. Synergistic effects by estradiol and v-H-ras were noted for three polypeptides. The secretion of Gcls-175 (Mr 50,000, pI 5.7) and Gcls-320 (Mr less than 14,000, pI 3.6, p-S2) was increased, while the secretion of Gcls-112 (Mr 76,000, pI 6.9) was decreased. Opposing effects of estradiol and v-H-ras were seen for seven polypeptides including the Mr 48,000 derivative of the Mr 52,000 protein (cathepsin D). These studies support the possibility that an extremely few, but specific polypeptides are regulated in association with quite diverse tumorigenic stimuli in MCF-7 human breast cancer cells.
Previous neuroanatomical studies in rats have demonstrated that the medial prefrontal cortex sends projections to the nucleus of the solitary tract which also receives the bulk of baroreceptor information from primary afferents within the IXth and Xth cranial nerves. The present study examines the influence of the prefrontal cortex on baroreceptor heart rate reflex in conscious rats. Baroreceptor reflex activity was examined in rats with bilateral excitotoxin (N-methyl-D-aspartate)-induced lesions of the medial prefrontal cortex and in control rats (artificial cerebrospinal fluid). Seventeen to eighteen days after lesioning, reflex heart rate responses were recorded following intravenous bolus doses of the pressor agent phenylephrine and the depressor agent sodium nitroprusside. Baroreceptor reflex parameters i.e., maximum and average baroreceptor reflex gain (or sensitivity): minimum and maximum heart rate plateaus; heart rate range; upper and lower reflex thresholds, were determined by sigmoidal computerized curve-fitting. Lesioning the medial prefrontal cortex did not affect resting mean arterial pressure and heart rate. However, the lesion reduced maximum and average baroreceptor reflex gain and produced a small reduction in lower reflex threshold. The other parameters were unaffected by the lesion. These observations suggest that although the medial prefrontal cortex does not exert a tonic influence on brainstem vasomotor neurons, there may be a descending excitatory projection from this brain region to medullary neurones involved in the baroreceptor reflex arc.