Ca2+ binding properties and Ca2(+)-dependent interactions of the isolated NH2-terminal alpha fragments of human complement proteases C1-r and C1-s

The NH2-terminal alpha fragments of human complement proteases C1-r and C1-s were obtained by limited proteolysis of the native proteins with trypsin, and isolated. C1-r alpha extended from residues 1 to 208 of C1-r A chain, with at least two cleavage sites within disulfide loops, after lysine 134 and arginine 202. C1-s alpha comprised residues 1-192 of the C1-s A chain, with one cleavage site within a disulfide loop, after arginine 186. C1-r alpha was monomeric either in the presence or absence of Ca2+ but formed Ca2(+)-dependent dimers with native C1-s. C1-s alpha dimerized in the presence of Ca2+ and formed Ca2(+)-dependent tetramers (C1-s alpha-C1-r-C1-r-C1-s alpha) with native C1-r. C1-r alpha and C1-s alpha associated in the presence of Ca2+ to form C1-r alpha-C1-s alpha heterodimers. Equilibrium dialysis studies indicated that each alpha region binds Ca2+ with a dissociation constant ranging from 19 microM (native proteins) to 38 microM (fragments). C1-r alpha, C1-r alpha-C1-s alpha, and the native C1-s-C1-r-C1-r-C1-s tetramer bound 0.9, 1.9, and 4.0 Ca2+ atoms/mol, respectively, whereas dimers C1-s alpha-C1-s alpha and C1-s-C1-s incorporated 2.9 and 3.0 Ca2+ atoms/mol. It is concluded that each alpha region contains one high affinity Ca2+ binding site. This 1:1 stoichiometry is maintained upon heterologous (C1-r-C1-s) interaction, whereas the homologous (C1-s-C1-s) interaction provides one additional binding site.     

Domain structure, stability, and interactions of human complement C1s-: characterization of a derivative lacking most of the B chain

A better understanding of the structure and function of C1 requires knowledge of the regions (domains) of the subcomponents that are responsible for Ca2+-dependent assembly. Toward this end, C1-s was digested with trypsin in the presence of Ca2+, a treatment that rapidly degraded the B chain, leaving a 56-kDa fragment comprised of a complete A chain disulfide linked to a small (less than 4-kDa) residual piece of the B chain. The purified fragment, referred to as C1-s-A, was shown by fast exclusion chromatography to be similar to C1-s in its ability to (1) reversibly dimerize in the presence of Ca2+, (2) substitute for C1-s in the formation of C1-r2-s2 tetramers, and (3) associate with C1-r and C1q to form macromolecular C1. Although C1-s-A was itself catalytically and hemolytically inactive, it competitively inhibited the expression of the hemolytic activity of C1-s in a reconstitution assay. When heated in the absence of Ca2+, C1-s exhibited a low-temperature transition (LTT) near 31 degrees C and a high-temperature transition (HTT) near 51 degrees C, similar to those previously observed in the homologous protein C1-r [Busby, T. F., & Ingham, K. C. (1987) Biochemistry 26, 5564-5571]. The midpoint of the LTT was shifted to 58 degrees C in 5 mM Ca2+ whereas the HTT was unaffected by Ca2+. C1-s-A exhibited only a LTT whose midpoint and Ca2+ dependence were similar to those of the LTT in C1-s. The HTT, which was accompanied by a loss of esterolytic activity, was reproduced in a plasmin-derived fragment representing the catalytic domain. These results provide strong support for the structural and functional independence of the catalytic and interaction domains of C1-s and strengthen current models regarding the role of these domains in various interactions. They also provide direct proof for the occurrence of Ca2+ binding sites on the A chain and demonstrate that all or most of the sites on C1-s that are responsible for its interaction with C1-r and C1q are located on the A chain.     

(p-Amidinophenyl)methanesulfonyl fluoride, an irreversible inhibitor of serine proteases

p-(Amidinophenyl)methanesulfonyl fluoride (p-APMSF) has been synthesized and shown to be a specific, irreversible inhibitor of the class of plasma serine proteases which demonstrate substrate specificity for the positively charged side chains of the amino acid lysine or arginine. In equimolar concentration, this compound causes immediate and complete irreversible inhibition of bovine trypsin and human thrombin. A 5-10-fold molar excess of reagent over enzyme is required to achieve complete irreversible inhibition of bovine Factor Xa, human plasmin, human C1-r, and human C1-s. the Ki of p-APMSF for all of the above-mentioned proteases is between 1 and 2 microM. In contrast, p-APMSF in large molar excess does not inactivate chymotrypsin or acetylcholinesterase. The unique reactivity of p-APMSF has been further shown in comparison with the related compound p-nitrophenyl (p-amidinophenyl)methanesulfonate which is an active-site titrant for thrombin but reacts poorly with Factor Xa, C1-r, and C1-s and is not hydrolyzed by bovine trypsin or human plasmin. Similarly, (p-amidinophenyl)methanesulfonate has a Ki of 30 microM for thrombin but is a poor inhibitor of trypsin, Factor Xa, C1-r, C1-s, and plasmin. Studies with bovine trypsin have demonstrated that the inhibitory activity of p-APMSF is the result of its interaction with the diisopropyl fluorophosphate reactive site. The unique reactivity of this inhibitor classifies it as one of the most effective active site directed reagents for this class of serine proteases. Collectively, these results suggest that the primary substrate binding site of these enzymes, which share a high degree of structural homology, do in fact significantly differ from each other in their ability to interact with low molecular weight inhibitors and synthetic substrates.     

Purification of a proteinase inhibitor from bovine serum with C1-inhibitor activity

This report describes the purification of a novel proteinase inhibitor from bovine serum. This protein was purified to apparent homogeneity employing affinity binding to sulfated dextran and precipitation by ammonium sulfate, followed by sequential chromatography on DEAE-cellulose, heparin-Sepharose and Sephacryl S-200. Quantitative enzyme-linked immunosorbent assays revealed that the concentration of this inhibitor is approximately 3 microM in bovine serum. The inhibitor is a single polypeptide chain with an estimated Mr of 83,000 as determined by SDS-polyacrylamide gel electrophoresis. An aspartic acid was found at the amino terminus of the protein; N-terminal amino acid sequence data indicated that there was no significant homology with other reported amino acid sequences. This bovine inhibitor covalently complexed the human proteinases C1-r, C1-s, factor XIIa and plasma kallikrein, which are also complexed and inactivated by human C1-inhibitor. In addition, the bovine inhibitor complexed and inactivated bovine chymotrypsin, a feature which functionally distinguishes it from human C1-inhibitor. Although the bovine inhibitor appears functionally very similar to C1-inhibitor, we found no evidence for structural homology with the human counterpart.     

Chemical and functional characterization of a fragment of C1-s containing the epidermal growth factor homology region

C1-s, one of the three subcomponents of C1-, the first component of complement, is a serine protease comprising two disulfide-linked chains, the B chain, containing the catalytic site, and the A chain, involved in Ca2+ binding and Ca2(+)-dependent interaction(s) with the other C1- subcomponents. In an attempt to identify the regions responsible for the latter functions, C1-s was submitted to limited proteolysis with plasmin, a treatment that split the A chain into three major fragments, alpha 1, alpha 2, and gamma. Fragment alpha 2, which comprised the epidermal growth factor-like (EGF-like) region of C1-s, was heterogeneous, starting at serine 97 or phenylalanine 105 and ending at lysine 195. This fragment was reduced and alkylated and then digested with elastase, and three peptides covering positions 131-135, 131-139, and 131-140 were characterized by amino acid analysis, Edman degradation, and mass spectrometry, showing that position 134 of C1-s is occupied partly by an asparagine (47%) and partly by an erythro-beta-hydroxyasparagine, in contrast with the homologous position (150) of C1-r which only contains erythro-beta-hydroxyasparagine. As measured by equilibrium dialysis, native alpha 2, like the other plasmin-cleavage fragments, did not retain the ability of intact C1-s to bind Ca2+. In the same way, plasmin cleavage abolished the ability of C1-s to dimerize or to associate with C1-r in the presence of Ca2+. In contrast, both alpha 2 and the N-terminal alpha 1 fragment, starting at serine 24 of the A chain, were able to compete significantly with intact C1s for the formation of the Ca2(+)-dependent C1-s-C1r-C1-r-C1-s tetramer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rates of dissociation of steroid and thyroid hormones from human serum albumin

A rapid filtration assay employing dextran-coated charcoal as acceptor particles for free hormone was used to measure rates of dissociation of steroid and thyroid hormones from human serum albumin. Modification of a previously described assay allowed measurements at 1-s intervals. Nevertheless, this still permitted only minimum estimates of the dissociation rate constants. The hormones studied were thyroxine, 3,5,3'-triiodothyronine, cortisol, corticosterone, testosterone, dihydrotestosterone, estradiol, progesterone, and aldosterone. The apparent dissociation rate constant of the thyroxine-albumin complex at 37 degrees C was 1.3 +/- 0.2 s-1 (t 1/2, 0.5 s). The apparent dissociation rate constants of the other hormone-albumin complexes at 37 degrees C generally exceeded 2 s-1 (t 1/2 less than 0.35 s). Apparent dissociation rate constants at 4 degrees C were only slightly lower. These findings indicate that steroid and thyroid hormones dissociate from albumin rapidly compared with the 1-s capillary transit times that characterize many tissues.     

Changes in protein conformation and stability accompany complex formation between human C1 inhibitor and C1-s

The fluorescence spectrum of C1 inhibitor (C1-Inh) in aqueous buffer has a maximum at 324 nm which shifts to 358 nm in 6.0 M guanidinium chloride (GdmC1), indicating that fluorescent tryptophans are buried in the native protein. When titrated with GdmC1, the fluorescence intensity, polarization, and emission maximum of C1-Inh and C1-s exhibited clear transitions which were more prominent than those of the enzyme-inhibitor complex. Two of the variables (intensity and emission maximum) suggest biphasic unfolding of C1-Inh. Differential absorption measurements and sodium iodide quenching of intrinsic fluorescence were consistent with a net increase in the exposure of tryptophans and tyrosines upon complex formation. This reaction, i.e., complex formation, was also accompanied by an increase in the ability to enhance the fluorescence of the hydrophobic probe 8-anilino-1-naphthalenesulfonate. Fluorescence assays of heat denaturation showed transitions at 40 and 52 degrees C for C1-s and at 60 degrees C for C1-Inh whereas there was no detectable melting transition for the complex. Similarly, differential scanning calorimetric measurements revealed transitions at 42, 52, and 62 degrees C for C1-s and one transition at 60 degrees C for C1-Inh, with no major transitions detectable for the complex. The ratio of the calorimetric enthalpy to the apparent van't Hoff enthalpy for thermal unfolding of C1-Inh was 1.6. Taken together, these results suggest that C1-Inh and C1-s are each composed of at least two independently unfolding domains and that complex formation, which involves conformational change, yields a protein substantially more stable than either component alone.     

Functional studies on the secreted form of human C4 (C4s), two incompletely processed two-subunit C4 molecules (beta - alpha + gamma and beta + alpha - gamma), and pro-C4

The functional properties of the secreted form of C4 (C4s), which has a Mr approximately 5000 greater than the predominant C4 molecule found in plasma (C4p), two incompletely processed two-chain C4 molecules (beta - alpha + gamma and beta + alpha - gamma), and the extracellular C4 precursor (designated pro-C4(E)] were evaluated. All four molecules are secreted in parallel by a human hepatoma-derived cell line (Hep G2). Secretion of hemolytically active C4 is linear up to approximately 12 hr, peaks at 24 hr, and then progressively decreases over the next 48 hr. This loss of C4s functional activity parallels the proteolytic conversion of C4s to C4bs. To compare the hemolytic efficiencies of C4s and C4p, a solid-phase competitive radioimmunoassay was developed to permit measurement of the small quantities of C4 antigen in these cultures. The hemolytic efficiencies of C4s and C4p were similar. These results indicate that extracellular processing of C4s to C4p does not modulate the hemolytic activity of the molecule. Consistent with their ability to bind methylamine, both the alpha s-chain and the alpha - gamma subunit undergo denaturation-induced autolysis. The extracellular and intracellular pro-C4 molecules are also sensitive to autolytic cleavage. Interestingly, the beta - alpha subunit is resistant to autolysis. In experiments in which C4s and C4p were cleaved by C1-s to C4bs, C4(beta - alpha + gamma), C4(beta + alpha - gamma), and pro-C4(E) were resistant to C1-s cleavage and thus hemolytically inactive relative to C4s. These data indicate that processing of C4 to a three-chain structure is required to provide the proper conformation for efficient activation by C1.     

A specific l-tri-iodothyronine-binding protein in the cytosol fraction of human breast adipose tissue

1. Binding of l-tri-[(125)I]iodothyronine to the cytosol fraction of normal human female breast adipose tissue was investigated by the charcoal adsorption method. Equilibrium of binding was reached after 120s at 25 degrees C. 2. The l-tri-[(125)I]iodothyronine-binding component is a protein; this was confirmed by experiments in which binding was totally lost after heating the cytosol fraction for 10min at 100 degrees C and in which binding was diminished after treatment with proteolytic enzymes and with thiol-group-blocking reagents. The binding protein was stable at -38 degrees C for several months. 3. It displayed saturability, high affinity (apparent K(d) 3.28nm) and a single class of binding sites. 4. High specificity for l-tri-iodothyronine and l-3,5-di-iodo-3'-isopropylthyronine was observed, whereas other iodothyronines were less effective in displacing l-tri-[(125)I]-iodothyronine from its binding site. 5. The binding of the hormone by the cytosol fraction did not show a pH optimum. 6. When cytosol fractions of adipose tissue from different females were subjected to radioimmunoassay for the determination of thyroxine-binding globulin a value of 0.304+/-0.11mug/mg of cytosol protein (mean+/-s.d., n=4) was obtained; the mean concentration in plasma was 0.309+/-0.07mug/mg of plasma protein (mean+/-s.d., n=3). 7. The K(a) value of 6.3x10(8)m(-1) of l-tri-[(125)I]iodothyronine for binding to plasma, the similar thermalinactivation profiles of binding and the reactivity to thiol-group-blocking reagents were some properties common between the binding components from the cytosol fraction and plasma. 8. These results suggest that the cytosol fraction of human female breast adipose tissue contains thyroxine-binding globulin; the protein that binds l-tri-[(125)I]iodothyronine with high affinity and specificity appears to be similar to thyroxine-binding globulin.     

Inhibition of human immunodeficiency virus type 1 activity in vitro by a new self-stabilized oligonucleotide with guanosine-thymidine quadruplex motifs

An oligonucleotide with a dimeric hairpin guanosine quadruplex (basket type structure) (dG3T4G3-s), containing phosphorothioate groups, was able to inhibit human immunodeficiency virus type 1 (HIV-1)-induced syncytium formation and virus production (as measured by p24 core antigen expression) in peripheral blood mononuclear cells. This oligonucleotide lacks primary sequence homology with the complementary (antisense) sequences to the HIV-1 genome. Furthermore, this oligonucleotide may have increased nuclease resistance. The activity of this oligonucleotide was increased when the phosphodiester backbone was replaced with a phosphorothioate backbone. In vivo results showed that dG3T4G3-s was capable of blocking the interaction between gp120 and CD4. We also found that dG3T4G3-s specifically inhibits the entry of T-cell line-tropic HIV-1 into cells. This compound is a viable candidate for evaluation as a therapeutic agent against HIV-1 in humans.     

Energy balance studies in frog skeletal muscles shortening at one-half maximal velocity

High-energy phosphate metabolism and energy liberated as heat and work were measured in 3-s tetani of frog sartorius muscle at 0 degree C. Two contraction periods were studied: (a) a 0.35-s period of shortening near half-maximum velocity beginning after 2 s of isometric stimulation, and (b) a 0.65-s isometric period immediately following the shortening. There were no significant changes in levels of ATP, ADP, or AMP in the two contraction periods. The observed changes in inorganic phosphate and creatine levels indicated that the only significant reaction occurring was phosphocreatine splitting. The mean rate of high-energy phosphate splitting during the shortening, 1.60 +/- 0.23 mumol X g-1 X s-1 (n = 24), was about fivefold higher than that in the 1-s period in the isometric tetanus, 0.32 +/- 0.11 mumol X g-1 X s- 1 (n = 17), observed in our previous study. The mean rate in the post- shortening period, 0.46 +/- 0.13 mumol X g-1 X s-1 (n = 17), was not significantly different from that in the 1-s period in the isometric tetanus. A large amount of heat plus work was produced during the shortening period, and this could be accounted for by simultaneous chemical changes. In the post-shortening period, the observed enthalpy was also accounted for by simultaneous chemical reactions. Thus, the present result is in sharp contrast to that obtained from a similar study performed at a shortening at Vmax, where an enthalpy excess was produced during shortening and an enthalpy deficit was produced during the period following the shortening.     

Myosin phosphorylation, twitch potentiation, and fatigue in human skeletal muscle

Twitch tension and phosphate incorporation into the phosphorylatable light chains (P-light chains) of myosin were studied during a 10-min recovery period following a 10- or 60-s maximal voluntary isometric contraction (MVC) in 18 subjects. Analysis of muscle biopsy samples obtained before, immediately after, 1 min, and 10 min following the 10-s MVC revealed that the 10-s MVC produced a modest but transient metabolic displacement from rest, a 35% decrease in phosphocreatine, and a threefold elevation in lactate concentration. Immediately after the 60-s MVC, ATP was decreased by 20%, phosphocreatine decreased by 84%, and lactate was elevated by 15-fold. Lactate remained elevated over the 10-min recovery period. Twitch force was maximally potentiated following the 10-s MVC and declined to rest by 10 min of recovery. Twitch force was 0.66 of rest value immediately after the 60-s MVC, then increased over the next 4 min to reach a potentiated value 21% greater than rest, before declining. Significant phosphate incorporation into P-light chains was observed immediately after both contractions, but dephosphorylation to rest values at the end of recovery was only noted for the 60-s condition. These results demonstrate an inconsistent relationship between twitch tension enhancement and P-light chain phosphorylation in the in vivo human model.     

Sulfated modification and cytotoxicity of <Emphasis Type="Italic">Portulaca oleracea</Emphasis> L. polysaccharides

A water-soluble polysaccharide (POP1) was isolated from Portulaca oleracea L. Four sulfated derivatives of POP1 (POP1-s1, POP1-s2, POP1-s3 and POP1-s4) were prepared by chlorosulfonic acid method with N,N-Dicyclohexylcarbodiimide (DCC) as a dehydration-condensation agent. FT-IR spectra and ¹³C NMR spectra indicated the sulfated groups had been introduced at the C-6 and C-2 positions of POP1. Sulfated derivatives had different degree of substitution (DS) ranging from 1.01 to 1.81, and different weight-average molecular mass (Mw) ranging from 41.4 to 48.5 KDa. Sulfated derivatives except POP1-s5 inhibited the growth of HepG2 cells and Hela cells in vitro significantly, which indicated that sulfated modification could enhance cytotoxicity of POP1 on tumor cells. Flow cytometric studies revealed that sulfated derivatives could mediate the cell-cycle arrest of Hela cells in the S phase.     

Torque potentiation and myosin light-chain phosphorylation in human muscle following a fatiguing contraction

Indices of electrically stimulated and maximal voluntary isometric muscle torgue and the phosphate content of myosin phosphorylatable light chains (P light chains) were studied during recovery following a 60-s maximal voluntary isometric contraction (MVC) in 21 human subjects. Analysis of muscle biopsy samples revealed that immediately after the 60-s MVC there were significant decreases in ATP (-15%) and phosphocreatine (-82%), and lactate concentration increased by 17-fold. All indices of muscle torque production were reduced by the 60-s MVC, but the twitch torque and torque at 10 Hz were relatively less reduced compared with the torque at 20 and 50 Hz or a 1-s MVC. Between 3 and 6 min of recovery, twitch torque and torque at 10 Hz stimulation were significantly potentiated, reaching peak values of 125 and 134%, respectively, compared with rest. Phosphate content of the fast and two slow P light chains was significantly increased over rest levels immediately after and 4 min after the 60-s MVC. These results suggest that myosin P light-chain phosphorylation could provide a mechanism to increase human muscle torque under conditions of submaximal contractile element activation following fatigue.     

Electrical and mechanical failures during sustained and intermittent contractions in humans

This paper compares the effects of sustained and intermittent contractions on electrical and mechanical failure during muscle fatigue in the human adductor pollicis electrically stimulated at 30 Hz via its motor nerve. Sixty-second sustained contractions are compared with a series of 60 1-s contractions, separated by 2.0-, 1.0-, and 0.5-s intervals for identical duration of tension development. Sixty-second sustained contractions decrease tetanic force to 60% (P less than 0.05) of initial values. No significant difference (P greater than 0.05) of force reduction was observed during intermittent 1-s contractions separated by 1-s intervals (-40%), but final force reduction was found to be significantly smaller (P less than 0.05) for 2-s intervals (-18%) and larger (P less than 0.05) for 0.5-s intervals (-65%). When identical force reduction is present in both fatigue tests, it appears that concomitant electrical failure is considerably different during sustained and intermittent contractions (P less than 0.05). This electromechanical dissociation suggests that slowing of conduction along nerve and muscle membranes, as well as possible increase of synaptic delay, does not explain the observed mechanical failure. It is therefore suggested that intracellular processes play the major role in contractile failure during sustained and intermittent contractions.     

A heteroglycan from the cyanobacterium Nostoc commune modulates LPS-induced inflammatory cytokine secretion by THP-1 monocytes through phosphorylation of ERK1/2 and Akt

Cyanobacteria (blue-green algae) have been consumed as food and used in folk medicine since ancient times to alleviate a variety of diseases. Cyanobacteria of the genus Nostoc have been shown to produce complex exopolysaccharides with antioxidant and antiviral activity. Furthermore, Nostoc sp. are common in cyanolichen symbiosis and lichen polysaccharides are known to have immunomodulating effects. Nc-5-s is a heteroglycan isolated from free-living colonies of Nostoc commune and its structure has been characterized in detail. The aim of this study was to determine the effects of Nc-5-s on the inflammatory response of lipopolysaccharide (LPS)-stimulated human THP-1 monocytes and how the effects are mediated. THP-1 monocytes primed with interferon-γ and stimulated with LPS in the presence of Nc-5-s secreted less of the pro-inflammatory cytokine interleukin (IL)-6 and more of the anti-inflammatory cytokine IL-10 than THP-1 monocytes stimulated without Nc-5-s. In contrast, Nc-5-s increased LPS-induced secretion of the pro-inflammatory cytokines tumor necrosis factor (TNF)-α and IL-8. Nc-5-s decreased LPS-induced phosphorylation of the extracellular regulated kinase (ERK)1/2 and Akt kinase, but did not affect phosphorylation of the p38 kinase, activation of the nuclear factor kappa B pathway, nor DNA binding of c-fos. These results show that Nc-5-s has anti-inflammatory effects on IL-6 and IL-10 secretion by THP-1 monocytes, but its effects are pro-inflammatory when it comes to TNF-α and IL-8. Furthermore, they show that the effects of Nc-5-s may be mediated through the ERK1/2 pathway and/or the Akt/phosphoinositide 3-kinase pathway and their downstream effectors. The ability of Nc-5-s to decrease IL-6 secretion, increase IL-10 secretion and moderate ERK1/2 activation indicates a potential for its development as an anti-inflammatory agent.     

Report of the First International Workshop on Human Chromosome 2 mapping 1991.

We report the isolation of a clone (pTR9) from a human chromosome 21 lambda phage library, which was found to contain two distinct components: (1) a previously unreported subfamily of human satellite III (pTR9-s3; 1,485 bp) and (2) an alpha satellite sequence (pTR9-alpha; 250 bp) containing 1.5 copies of a 171-bp alphoid unit that shows 88.4% homology to a previously reported alpha satellite consensus sequence. The two components are separated by two direct repeats of 9 bp. Use of the polymerase chain reaction (PCR) to amplify across the junction between pTR9-s3 and pTR9-alpha established that these two sequences are contiguous in total human genomic DNA and in DNA derived from somatic cell hybrids carrying human chromosomes 13, 14, or 21. A related, but considerably more diverged, sequence was also detected on chromosome 15. Southern analysis of somatic cell hybrids at high stringency revealed a common structure of the pTR9-s3 sequence on chromosomes 13, 14, and 21 but not on 15 or 22. This sequence should be useful for the study of the structural organisation of the centromere of these chromosomes and the mechanism of their involvement in Robertsonian translocations.     

Differential acquisition of specific components of a classically conditioned arterial blood pressure response in rat

Presenting a 15-s pulsed tone, the conditional stimulus (CS(+)), followed by 0.5-s tail shock, to a well-trained rat causes a sudden, but transient, pressor response (C(1)). Blood pressure (BP) then drops before increasing again (C(2)). A steady tone of the same frequency never followed by a shock (a discriminative stimulus, or CS(-)) evokes a C(1) but not a C(2) response. Experiment 1 tested the hypothesis that this BP response pattern does not depend on the nature of the tone (i.e., pulsed vs. steady) used for CS(+) and CS(-). The tones were reversed from the traditional paradigm, above, in nine rats. The C(1) BP increase for a steady-tone CS(+) (+4.8 +/- 1.9 mmHg, mean change +/- SE) and a pulsed CS(-) (+2.9 +/- 1.3 mmHg) did not differ. Conversely, C(2) showed a clear discrimination (CS(+): +5.1 +/- 1.2 mmHg, CS(-): +0 .7 +/- 0.8 mmHg; P < 0.05). Experiment 2 tested the hypothesis that the C(1) and C(2) BP responses first appear at different times during training. On training day 1, five 15-s pulsed tones (CS(+)) were presented to each of 18 rats; the last tone was followed by a tail shock. Likewise, five steady CS(-) tones never followed by shock were given. Training continued for 2 more days, with each CS(+) followed by shock. At the end of day 2, CS(+) evoked a C(1) BP response (+3.9 +/- 0.9 mmHg) but no C(2) (+0.6 +/- 0.4 mmHg, not significant vs. pretone). By the end of day 3, CS(+) evoked a significant (vs. baseline) C(1) (+7.3 +/- 1.4 mmHg) and C(2) (+3.3 +/- 0.8 mmHg). Conversely, although CS(-) evoked a C(1) response (3.5 +/- 1.3 mmHg), there was no C(2) (+0.7 +/- 0.5 mmHg; not significant). We conclude that 1) C(1) and C(2) are acquired at different rates, 2) early in training C(1) is an orienting response evoked by both tones, and 3) C(2) is only acquired as an animal learns to associate the CS(+) tone with shock. This suggests that C(1) and C(2) are controlled by different processes in the brain.