Biological evaluation of recombinant human erythropoietin in pharmaceutical products

The potencies of mammalian cell-derived recombinant human erythropoietin pharmaceutical preparations, from a total of five manufacturers, were assessed by in vivo bioassay using standardized protocols. Eight-week-old normocythemic mice received a single subcutaneous injection followed by blood sampling 96 h later or multiple daily injections with blood sampling 24 h after the last injection. Reticulocyte counting by microscopic examination was employed as the end-point using the brilliant cresyl blue or selective hemolysis methods, together with automated flow cytometry. Different injection schedules were investigated and dose-response curves for the European Pharmacopoeia Biological Reference Preparation of erythropoietin were compared. Manual and automated methods of reticulocyte counting were correlated with respect to assay validity and precision. Using 8 mice per treatment group, intra-assay precision determined for all of the assays in the study showed coefficients of variation of 12.1-28.4% for the brilliant cresyl blue method, 14.1-30.8% for the selective hemolysis method and 8.5-19.7% for the flow cytometry method. Applying the single injection protocol, a combination of at least two independent assays was required to achieve the precision potency and confidence limits indicated by the manufacturers, while the multiple daily injection protocol yielded the same acceptable results within a single assay. Although the latter protocol using flow cytometry for reticulocyte counting gave more precise and reproducible results (intra-assay coefficients of variation: 5.9-14.2%), the well-characterized manual methods provide equally valid alternatives for the quality control of recombinant human erythropoietin therapeutic products.

Genotoxic, neurotoxic and neuroprotective activities of apomorphine and its oxidized derivative 8-oxo-apomorphine

Apomorphine is a dopamine receptor agonist proposed to be a neuroprotective agent in the treatment of patients with Parkinson's disease. Both in vivo and in vitro studies have shown that apomorphine displays both antioxidant and pro-oxidant actions, and might have either neuroprotective or neurotoxic effects on the central nervous system. Some of the neurotoxic effects of apomorphine are mediated by its oxidation derivatives. In the present review, we discuss recent studies from our laboratory in which the molecular, cellular and neurobehavioral effects of apomorphine and its oxidized derivative, 8-oxo-apomorphine-semiquinone (8-OASQ), were evaluated in different experimental models, i.e., in vitro genotoxicity in Salmonella/microsome assay and WP2 Mutoxitest, sensitivity assay in Saccharomyces cerevisiae, neurobehavioral procedures (inhibition avoidance task, open field behavior, and habituation) in rats, stereotyped behavior in mice, and Comet assay and oxidative stress analyses in mouse brain. Our results show that apomorphine and 8-OASQ induce differential mutagenic, neurochemical and neurobehavioral effects. 8-OASQ displays cytotoxic effects and oxidative and frameshift mutagenic activities, while apomorphine shows antimutagenic and antioxidant effects in vitro. 8-OASQ induces a significant increase of DNA damage in mouse brain tissue. Both apomorphine and 8-OASQ impair memory for aversive training in rats, although the two drugs showed a different dose-response pattern. 8-OASQ fails to induce stereotyped behaviors in mice. The implications of these findings are discussed in the light of evidence from studies by other groups. We propose that the neuroprotective and neurotoxic effects of dopamine agonists might be mediated, in part, by their oxidized metabolites.

Indole ring oxidation by activated leukocytes prevents the production of hypochlorous acid

Hypochlorous acid (HOCl) released by activated leukocytes has been implicated in the tissue damage that characterizes chronic inflammatory diseases. In this investigation, 14 indole derivatives, including metabolites such as melatonin, tryptophan and indole-3-acetic acid, were screened for their ability to inhibit the generation of this endogenous oxidant by stimulated leukocytes. The release of HOCl was measured by the production of taurine-chloramine when the leukocytes (2 x 10(6) cells/mL) were incubated at 37ºC in 10 mM phosphate-buffered saline, pH 7.4, for 30 min with 5 mM taurine and stimulated with 100 nM phorbol-12-myristate acetate. Irrespective of the group substituted in the indole ring, all the compounds tested including indole, 2-methylindole, 3-methylindole, 2,3-dimethylindole, 2,5-dimethylindole, 2-phenylindole, 5-methoxyindole, 6-methoxyindole, 5-methoxy-2-methylindole, melatonin, tryptophan, indole-3-acetic acid, 5-methoxy-2-methyl-3-indole-acetic acid, and indomethacin (10 µM) inhibited the chlorinating activity of myeloperoxidase (MPO) in the 23-72% range. The compounds 3-methylindole and indole-3-acetic acid were chosen as representative of indole derivatives in a dose-response study using purified MPO. The IC50 obtained were 0.10 ± 0.03 and 5.0 ± 1.0 µM (N = 13), respectively. These compounds did not affect the peroxidation activity of MPO or the production of superoxide anion by stimulated leukocytes. By following the spectral change of MPO during the enzyme turnover, the inhibition of HOCl production can be explained on the basis of the accumulation of the redox form compound-II (MPO-II), which is an inactive chlorinating species. These results show that indole derivatives are effective and selective inhibitors of MPO-chlorinating activity.

Adenosine A1 receptor-mediated inhibition of in vitro prolactin secretion from the rat anterior pituitary

In previous studies, we demonstrated biphasic purinergic effects on prolactin (PRL) secretion stimulated by an adenosine A2 agonist. In the present study, we investigated the role of the activation of adenosine A1 receptors by (R)-N6-(2-phenylisopropyl)adenosine (R-PIA) at the pituitary level in in vitro PRL secretion. Hemipituitaries (one per cuvette in five replicates) from adult male rats were incubated. Administration of R-PIA (0.001, 0.01, 0.1, 1, and 10 µM) induced a reduction of PRL secretion into the medium in a U-shaped dose-response curve. The maximal reduction was obtained with 0.1 µM R-PIA (mean ± SEM, 36.01 ± 5.53 ng/mg tissue weight (t.w.)) treatment compared to control (264.56 ± 15.46 ng/mg t.w.). R-PIA inhibition (0.01 µM = 141.97 ± 15.79 vs control = 244.77 ± 13.79 ng/mg t.w.) of PRL release was blocked by 1 µM cyclopentyltheophylline, a specific A1 receptor antagonist (1 µM = 212.360 ± 26.560 ng/mg t.w.), whereas cyclopentyltheophylline alone (0.01, 0.1, 1 µM) had no effect. R-PIA (0.001, 0.01, 0.1, 1 µM) produced inhibition of PRL secretion stimulated by both phospholipase C (0.5 IU/mL; 977.44 ± 76.17 ng/mg t.w.) and dibutyryl cAMP (1 mM; 415.93 ± 37.66 ng/mg t.w.) with nadir established at the dose of 0.1 µM (225.55 ± 71.42 and 201.9 ± 19.08 ng/mg t.w., respectively). Similarly, R-PIA (0.01 µM) decreased (242.00 ± 24.00 ng/mg t.w.) the PRL secretion stimulated by cholera toxin (0.5 mg/mL; 1050.00 ± 70.00 ng/mg t.w.). In contrast, R-PIA had no effect (468.00 ± 34.00 ng/mg t.w.) on PRL secretion stimulation by pertussis toxin (0.5 mg/mL; 430.00 ± 26.00 ng/mg t.w.). These results suggest that inhibition of PRL secretion after A1 receptor activation by R-PIA is mediated by a Gi protein-dependent mechanism.

Effects of a methanolic fraction of soybean seeds on the transcriptional activity of peroxisome proliferator-activated receptors (PPAR)

Since the anti-inflammatory, antidiabetic and hypolipidemic effects of soy isoflavones may be mediated by activation of peroxisome proliferator-activated receptors (PPAR), the present study investigated whether the methanolic fractions obtained from soybean seeds (E1) and soybean seed coats with hypocotyls (E2) could influence PPARα, PPARγ and PPARβ/δ transcriptional activity. The isoflavones from E1 and E2 were quantified by HPLC analysis. E1 and E2 were rich in isoflavones (daidzin, glycitin, genistin, malonyldaidzin, malonylglycitin, malonylgenistin, daidzein, glycitein, and genistein). Moreover, E1 and E2 showed no evidence of genetically modified material containing the gene CP4 EPSPS. To investigate PPAR transcriptional activity, human promonocytic U-937 cells were treated with E1 and E2 (200, 400, 800, and 1600 µg/mL), positive controls or vehicle. Data are reported as fold-activation of the luciferase reporter driven by the PPAR-responsive element. Dose-response analysis revealed that E1 and E2 induced the transcriptional activity of PPARα (P < 0.001), with activation comparable to that obtained with 0.1 mM bezafibrate (positive control) at 1600 µg/mL (4-fold) and 800 µg/mL (9-fold), respectively. In addition, dose-response analysis revealed that E1 and E2 activated PPARβ/δ (P < 0.05), and the activation at 800 µg/mL (4- and 9-fold, respectively) was comparable to that of 0.1 mM bezafibrate (positive control). However, no effect on PPARγ was observed. Activation of PPARα is consistent with the lipid-lowering activity of soy isoflavones in vivo, but further studies are needed to determine the physiological significance of PPARβ/δ activation.

Consequences of cerebroventricular insulin injection on renal sodium handling in rats: effect of inhibition of central nitric oxide synthase

In the present study, we investigated the effects of acute intracerebroventricular (icv) insulin administration on central mechanisms regulating urinary sodium excretion in simultaneously centrally NG-nitro-L-arginine methylester (L-NAME)-injected unanesthetized rats. Male Wistar-Hannover rats were randomly assigned to one of five groups: a) icv 0.15 M NaCl-injected rats (control, N = 10), b) icv dose-response (1.26, 12.6 and 126 ng/3 µL) insulin-injected rats (N = 10), c) rats icv injected with 60 µg L-NAME in combination with NaCl (N = 10) or d) with insulin (N = 10), and e) subcutaneously insulin-injected rats (N = 5). Centrally administered insulin produced an increase in urinary output of sodium (NaCl: 855.6 ± 85.1 Δ%/min; 126 ng insulin: 2055 ± 310.6 Δ%/min; P = 0.005) and potassium (NaCl: 460.4 ± 100 Δ%/min; 126 ng insulin: 669.2 ± 60.8 Δ%/min; P = 0.025). The urinary sodium excretion response to icv 126 ng insulin microinjection was significantly attenuated by combined administration of L-NAME (126 ng insulin: 1935 ± 258.3 Δ%/min; L-NAME + 126 ng insulin: 582.3 ± 69.6 Δ%/min; P = 0.01). Insulin-induced natriuresis occurred by increasing post-proximal sodium excretion, despite an unchanged glomerular filtration rate. Although the rationale for decreased urinary sodium excretion induced by combined icv L-NAME and insulin administration is unknown, it is tempting to suggest that perhaps one of the efferent signals triggered by insulin in the CNS may be nitrergic in nature.

Characterization of angiotensin-converting enzymes 1 and 2 in the soleus and plantaris muscles of rats

Angiotensin-converting enzymes 1 (ACE1) and 2 (ACE2) are key enzymes of the renin-angiotensin system, which act antagonistically to regulate the levels of angiotensin II (Ang II) and Ang-(1-7). Considerable data show that ACE1 acts on normal skeletal muscle functions and architecture. However, little is known about ACE1 levels in muscles with different fiber compositions. Furthermore, ACE2 levels in skeletal muscle are not known. Therefore, the purpose of this study was to characterize protein expression and ACE1 and ACE2 activities in the soleus and plantaris muscles. Eight-week-old female Wistar rats (N = 8) were killed by decapitation and the muscle tissues harvested for biochemical and molecular analyses. ACE1 and ACE2 activities were investigated by a fluorometric method using Abz-FRK(Dnp)P-OH and Mca-YVADAPK(Dnp)-OH fluorogenic substrates, respectively. ACE1 and ACE2 protein expression was analyzed by Western blot. ACE2 was expressed in the skeletal muscle of rats. There was no difference between the soleus (type I) and plantaris (type II) muscles in terms of ACE2 activity (17.35 ± 1.7 vs 15.09 ± 0.8 uF·min-1·mg-1, respectively) and protein expression. ACE1 activity was higher in the plantaris muscle than in the soleus (71.5 ± 3.9 vs 57.9 ± 1.1 uF·min-1·mg-1, respectively). Moreover, a comparative dose-response curve of protein expression was established in the soleus and plantaris muscles, which indicated higher ACE1 levels in the plantaris muscle. The present findings showed similar ACE2 levels in the soleus and plantaris muscles that might result in a similar Ang II response; however, lower ACE1 levels could attenuate Ang II production and reduce bradykinin degradation in the soleus muscle compared to the plantaris. These effects should enhance the aerobic capacity necessary for oxidative muscle activity.

Functional changes of dendritic cells in hypersensivity reactions to amoxicillin

A better understanding of dendritic cell (DC) involvement in responses to haptenic drugs is needed, because it represents a possible approach to the development of an in vitro test, which could identify patients prone to drug allergies. There are two main DC subsets: plasmacytoid DC (pDC) and myeloid DC (mDC). β-lactams form hapten-carrier conjugates and may provide a suitable model to study DC behavior in drug allergy reactions. It has been demonstrated that drugs interact differently with DC in drug allergic and non-allergic patients, but there are no studies regarding these subsets. Our aim was to assess the functional changes of mDC and pDC harvested from an amoxicillin-hypersensitive 32-year-old woman who experienced a severe maculopapular exanthema as reflected in interleukin-6 (IL-6) production after stimulation with this drug and penicillin. We also aim to demonstrate, for the first time, the feasibility of this method for dendritic cell isolation followed by in vitro stimulation for studies of drug allergy physiopathology. DC were harvested using a double Percoll density gradient, which generates a basophil-depleted cell (BDC) suspension. Further, pDC were isolated by blood DC antigen 4-positive magnetic selection and gravity filtration through magnetized columns. After stimulation with amoxicillin, penicillin and positive and negative controls, IL-6 production was measured by ELISA. A positive dose-response curve for IL-6 after stimulation with amoxicillin and penicillin was observed for pDC, but not for mDC or BDC suspension. These preliminary results demonstrate the feasibility of this methodology to expand the knowledge of the effect of dendritic cell activation by drug allergens.

Fine-tuning of defensive behaviors in the dorsal periaqueductal gray by atypical neurotransmitters

This paper presents an up-to-date review of the evidence indicating that atypical neurotransmitters such as nitric oxide (NO) and endocannabinoids (eCBs) play an important role in the regulation of aversive responses in the periaqueductal gray (PAG). Among the results supporting this role, several studies have shown that inhibitors of neuronal NO synthase or cannabinoid receptor type 1 (CB1) receptor agonists cause clear anxiolytic responses when injected into this region. The nitrergic and eCB systems can regulate the activity of classical neurotransmitters such as glutamate and γ-aminobutyric acid (GABA) that control PAG activity. We propose that they exert a ‘fine-tuning’ regulatory control of defensive responses in this area. This control, however, is probably complex, which may explain the usually bell-shaped dose-response curves observed with drugs that act on NO- or CB1-mediated neurotransmission. Even if the mechanisms responsible for this complex interaction are still poorly understood, they are beginning to be recognized. For example, activation of transient receptor potential vanilloid type-1 channel (TRPV1) receptors by anandamide seems to counteract the anxiolytic effects induced by CB1 receptor activation caused by this compound. Further studies, however, are needed to identify other mechanisms responsible for this fine-tuning effect.

Assessment of indicators of vitamin A status in non-cirrhotic chronic hepatitis C patients

Subjects with chronic liver disease are susceptible to hypovitaminosis A due to several factors. Therefore, identifying patients with vitamin deficiency and a requirement for vitamin supplementation is important. Most studies assessing vitamin A in the context of hepatic disorders are conducted using cirrhotic patients. A cross-sectional study was conducted in 43 non-cirrhotic patients with chronic hepatitis C to evaluate markers of vitamin A status represented by serum retinol, liver retinol, and serum retinol-binding protein levels. We also performed the relative dose-response test, which provides an indirect estimate of hepatic vitamin A reserves. These vitamin A indicators were assessed according to the stage of liver fibrosis using the METAVIR score and the body mass index. The sample study was predominantly composed of male subjects (63%) with mild liver fibrosis (F1). The relative dose-response test was <20% in all subjects, indicating vitamin A sufficiency. Overweight or obese patients had higher serum retinol levels than those with a normal body mass index (2.6 and 1.9 µmol/L, respectively; P<0.01). Subjects with moderate liver fibrosis (F2) showed lower levels of serum retinol (1.9 vs 2.5 µmol/L, P=0.01) and retinol-binding protein levels compared with those with mild fibrosis (F1) (46.3 vs 67.7 µg/mL, P<0.01). These results suggested an effect of being overweight on serum retinol levels. Furthermore, more advanced stages of liver fibrosis were related to a decrease in serum vitamin A levels.

Acute but not chronic metabolic acidosis potentiates the acetylcholine-induced reduction in blood pressure: an endothelium-dependent effect

Metabolic acidosis has profound effects on vascular tone. This study investigated the in vivo effects of acute metabolic acidosis (AMA) and chronic metabolic acidosis (CMA) on hemodynamic parameters and endothelial function. CMA was induced by ad libitum intake of 1% NH4Cl for 7 days, and AMA was induced by a 3-h infusion of 6 M NH4Cl (1 mL/kg, diluted 1:10). Phenylephrine (Phe) and acetylcholine (Ach) dose-response curves were performed by venous infusion with simultaneous venous and arterial blood pressure monitoring. Plasma nitrite/nitrate (NOx) was measured by chemiluminescence. The CMA group had a blood pH of 7.15±0.03, which was associated with reduced bicarbonate (13.8±0.98 mmol/L) and no change in the partial pressure of arterial carbon dioxide (PaCO2). The AMA group had a pH of 7.20±0.01, which was associated with decreases in bicarbonate (10.8±0.54 mmol/L) and PaCO2 (47.8±2.54 to 23.2±0.74 mmHg) and accompanied by hyperventilation. Phe or ACh infusion did not affect arterial or venous blood pressure in the CMA group. However, the ACh infusion decreased the arterial blood pressure (ΔBP: -28.0±2.35 mm Hg [AMA] to -4.5±2.89 mmHg [control]) in the AMA group. Plasma NOx was normal after CMA but increased after AMA (25.3±0.88 to 31.3±0.54 μM). These results indicate that AMA, but not CMA, potentiated the Ach-induced decrease in blood pressure and led to an increase in plasma NOx, reinforcing the effect of pH imbalance on vascular tone and blood pressure control.

Development and chemical characterization of flour obtained from the external mesocarp of "pequizeiro" fruit

The objective of this work was to develop a recommendation for the chemical peeling of pequi fruit and characterize the flour obtained from the external mesocarp of "Pequizeiro", pequi tree (Caryocar brasiliense Camb.). The technology applied to obtain the external mesocarp pequi flour included the epicarp removal with NaOH solution. The Response Surface Method was used to optimize the chemical peeling process by applying the Central Composite Rotatable Design, with eleven trials including three replicates at the central point, varying the NaOH aqueous solution concentration and fruit immersion time. The mass loss was evaluated through the analysis of variance and using bi and three dimensional graphs. The chemical characteristics of the external mesocarp pequi flour evaluated were: moisture content, ashes, proteins, lipids, total carbohydrates, dietary fiber, and some minerals. The best combination for an efficient removal of the fruit peel with the lowest mass loss was reached with 7.05 minutes of immersion in a 5.08 g.L-1 NaOH aqueous solution. This study indicated that the external mesocarp pequi flour is a food source rich in dietary fiber, carbohydrates, ashes, magnesium, calcium, manganese, and copper, but it is poor in lipids, zinc, and iron.

Assessment of toxic potential of Cerrado fruit seeds using Artemia salina bioassay

Artemia salina bioassay was used to assess toxicity of seeds and kernels of Brazilian fruits from cerrado (central high plains region) and other inner regions of the country. Water extracts of the kernels were filtered and added to Artemia cultures containing ten individuals per mL. Dose - response curves were constructed, and LD50 values were calculated. Pure potassium cyanide standard was used to draw a calibration curve for comparison to detect the presence of cyanide in the samples tested. Extracts of the seeds of araticum, mangaba, cagaita, jatobá, and tucumã were found toxic to Artemia salina, and some of the dose - response curves were very similar in shape to those obtained with pure potassium cyanide standards, while the samples of baru, cajá-manga, siriguela, trauma, and veludo showed no toxicity at all. The Guignard test, specific for cyanide detection, showed negative results in all toxic samples, suggesting the presence of other toxic compounds rather than cyanide. The comparison of araticum dose - response curve with those of other annonaceous fruits suggests the presence acetogenins as the main toxic compounds in the seeds. These results could be useful to prevent poisoning by industrial derivatives of the fruits studied.

The role of daytime napping in sleepiness and cognitive function in 24-70 year olds /

Daytime napping improves well-being and performance for young adults. The benefits of napping in older adults should be investigated because they have fragmented nocturnal sleep, cognitive declines, and more opportunity to nap. In addition, experience with napping might influence the benefits of napping. Study 1 examined the role of experience with napping in young adults. Habitual (n = 23) and non-habitual nappers (n = 16) were randomly assigned to a 20-minute nap or a 20- minute reading condition. Both groups slept the same according to macro architecture. However, microarchitecture showed greater theta, alpha, and beta power during Stage 1, and greater delta, alpha, and sigma power during Stage 2 for habitual nappers, for the most part indicating better sleep. Both groups felt less sleepy after the nap. P2 latency, reflecting information processing, decreased after the nap for habitual nappers, and after the control condition for non-habitual nappers. In sum, both groups who slept felt better, but only the habitual nappers who napped gained a benefit in terms of information processing. Based on this outcome, experience with napping was investigated in Study 2. Study 2 examined the extent to which daytime napping enhanced cognition in older adults, especially frontal lobe function. Cognitive deficits in older adults may be due to sleep loss and age-related decline in brain functioning. Longer naps were expected to provide greater improvement, particularly for older adults, by reducing sleep pressure. Thirty-two adults, aged 24-70 years, participated in a repeated measures dose-response manipulation of sleep pressure. Twenty- and sixty-minute naps were compared to a no-nap condition in three age groups. Mood, subjective sleepiness, reaction time, working memory, 11 novelty detection, and waking electro physiological measures were taken before and after each condition. EEG was also recorded during each nap or rest condition. Napping reduced subjective sleepiness, improved working memory (serial addition / subtraction task), and improved attention (reduced P2 amplitude). Physiological sleepiness (i.e., waking theta power) increased following the control condition, and decreased after the longer nap. Increased beta power after the short nap, and seen with older adults overall, may have reflected increased mental effort. Older adults had longer latencies and smaller amplitudes for several event-related potential components, and higher beta and gamma power. Following the longer nap, gamma power decreased for older adults, but increased for young adults. Beta and gamma power may represent enhanced alertness or mental effort. In addition, Nl amplitude showed that benefits depend on the preceding nap length as well as age. Since the middle group had smaller Nl amplitudes following the short nap and rest condition, it is possible that they needed a longer nap to maintain alertness. Older adults did not show improvements to Nl amplitude following any condition; they may have needed a nap longer than 60 minutes to gain benefits to attention or early information processing. Sleep characteristics were not related to benefits of napping. Experience with napping was also investigated. Subjective data confirmed habitual nappers were happier to nap, while non-habitual nappers were happier to stay awake, reflecting self-identified napping habits. Non-habitual nappers were sleepier after a nap, and had faster brain activity (i.e., heightened vigilance) at sleep onset. These reasons may explain why non-habitual nappers choose not to nap.

Effects of low doses of quinpirole on production of 50 kHz vocalizations in Wistar rats

Rats emit two distinct types of ultrasonic vocalizations in adulthood: 22 kHz (aversive situation), and 50 kHz calls (appetitive situation). The present project is focussed on pharmacological studies of 50 kHz vocalizations. The 50 kHz calls are elicited from dopaminergic activation in the meso limbic pathway and are emitted in such appetitive situations as social contact(s), sexual encounters, food reward, etc. Eighty-five male rats were stereotaxically implanted with bilateral guide cannulae in the nucleus accumbens shell (A= 9.7, L= 1.2, V= 6.7). Quinpirole, a D2/D3 dopaminergic agonist, was injected in low doses to the nucleus accumbens shell in an attempt to elicit 50 kHz vocalizations. A dose response was obtained for the low dose range of quinpirole for six doses: 0.025 Jlg, 0.06 Jlg, 0.12 Jlg, 0.25 Jlg, 0.5 Jlg, and 1.0 Jlg. It was found that only application of the 0.25 Jlg dose of quinpirole and the 7 Jlg dose of amphetamine (positive control) significantly increased the total number of 50 kHz calls (p < 0.006 and p < 0.004 respectively); and particularly significantly increased the frequency modulated type of these calls (p < 0.01, and p < 0.006 respectively). In a double injection procedure, the dose of 0.25 Jlg quinpirole was antagonized with raclopride (D2 antagonist) or U99194A maleate (D3 antagonist) in an attempt to antagonize the response. The 0.25 Jlg dose of quinpirole was successfully antagonized by pre-treatment with an equimolar dose of U99194A maleate (p < 0.008) but not with raclopride. The 7Jlg amphetamine response was also antagonized with an equimolar dose of raclopride. Based on these results, it seems that low doses of quinpirole, particularly the 0.25 Jlg dose, are capable of increasing 50 kHz vocalizations in rats and do so by activation of the D3 dopamine receptor. This is not a biphasic response as seen with locomotor studies. Also noteworthy is the increase in frequency modulated 50 kHz calls elicited by the 0.25 Jlg dose of quinpirole indicating a possible increase in positive affect.